Amphiphilic compounds with neuroprotective properties

ABSTRACT

Amphiphilic compounds with tetradecahydrophenanthrene skeleton and their enantiomers, exhibiting neuroprotective effects, their use in methods of treatment of neuropsychiatric disorders associated with an imbalance in glutamatergic neurotransmitter system, such as ischemic damage of CNS, neurodegenerative changes and disorders of CNS, affective disorders, depression, post-traumatic stress disorder and diseases related to stress, anxiety, schizophrenia and psychotic disorders, pain, addiction, multiple sclerosis, epilepsy, glioma, and a pharmaceutical composition containing compound.

FIELD OF INVENTION

The present invention is in the field of pharmaceutical chemistry. Theobjective is a set of compounds which inhibit excessively activatedmodulate NMDA receptors, and thus protect the tissue of the centralnervous system (CNS) against excessive action of glutamate.

BACKGROUND ART

NMDA receptors are multiprotein tetrameric complexes, which are composedof two NR1 subunits and two NR2A-2D subunits that form the ion channelfor positive ions (Nature 438, 185-192 (2005)).

Glutamate is the major excitatory neurotransmitter in the centralnervous system of mammals. Responses of the post synaptic neuron aregenerated during synaptic transmission via ionotropic and metabotropicglutamate receptors. N-Methyl-D-aspartate receptors (NMDA), AMPA andkainite receptors belong to the family of ionotropic glutamatereceptors.

Although current evidence suggests the role of different subtypes ofglutamate receptors in glutamate induced excitotoxicity, ionotropicreceptors are considered to be a key player in these processes.Activation of ionotropic glutamate receptors leads to changes inintracellular concentration of ions, especially calcium and sodium.Toxicity of higher levels of glutamate is generally associated with anincrease in intracellular Ca²⁺ levels. Currently, it is relatively wellestablished that there is a direct relationship between the excessiveinflux of calcium into the cells and glutamate-induced neuronal damage.Glutamate-induced pathological increase in intracellular calcium isattributed to prolonged activation of ionotropic glutamate receptors.Increases in intracellular calcium may trigger a cascade ofneurotoxicity.

A number of preclinical studies have documented striking ability of NMDAantagonists to prevent an excessive action of glutamate on nerve cellsand thereby reduce the impairment of the function of CNS. However, fromthe clinical point of view their neuroprotective potential is small. Dueto the fact that NMDA receptors are one of the most widespread types ofreceptors in the CNS, their administration lead usually to a number ofserious side effects (e.g. distortion, induction of motoric psychoses ofthe schizophrenic type, etc.).

On the other hand, a great variety of NMDA receptors and their differentdistribution at synapses and in the brain and various functional statesof this receptor offers great possibility of seeking for agents thatselectively affect only a specific group of NMDA receptors and therebyreduce the occurrence of unanticipated and undesirable effects whilemaintaining the neuroprotective activity (Pharmacol. Rev. 51, 7-61(1999); Semin. Cell Dev. Biol. 17, 592-604 (2006); Top. Med. Chem. 6,749-770 (2006); Anesth. Analg. 97, 1108-1116 (2003); Curr. Opin.Pharmacol. 6, 53-60 (2006); Curr. Opin. Investig. Drugs 4, 826-832(2003).

Previous results showed that naturally occurring3alpha,5beta-pregnanolone sulfate affects the activity of NMDA receptorsby the use-dependent manner. Due to this mechanism of action,pregnanolone sulfate has pronounced inhibitory action on NMDA receptorstonically activated by glutamate than phasically activated NMDAreceptors during synaptic transmission. The activation of tonicallyactivated extrasynaptic NMDA receptors is essential for the excitotoxicaction of glutamate (J. Neurosci. 25, 8439-50 (2005)).

Therefore, we have started a development and testing of novel NMDAantagonists derived from neurosteroids. These newly synthesizedcompounds exhibit affinity for extrasynaptic NMDA receptors. Moreover,our previous electrophysiological studies have shown that this type ofcompounds binds only to the long-term opened-NMDA receptors. Thesupposed mechanism of the neuroprotective effect is blocking ofexcessive penetration of calcium into cells through the open NMDAreceptors. As these compounds do not have affinity to other types ofNMDA receptors, it is believed that they would minimally affect thesignal transmission between neurons.

In the last decade, the biomedical research has been focused on researchof the role of neurosteroids in the pathophysiology of manyneuropsychiatric disorders and to assess therapeutic potential of thesecompounds. Mechanism of action of neurosteroids is associated with theiractivity on the NMDA and GABA receptors. Experimental studies withanimal models suggest potential of neurosteroids to treat a variety ofcentral nervous disorders, particularly neurodegenerative diseases,multiple sclerosis, affective disorders, alcoholism, pain, insomnia orschizophrenia (J. Pharm. Exp. Ther. 116, 1-6 (2007); J. Pharm. Exp.Ther. 293, 747 (2000)).

Neurosteroids play a crucial role in the regulation of stress and therelated CNS disorders. The level of neurosteroids temporarily afterexposure to stress increases, as it is an adaptive mechanism. Oncontrary, experimental models of chronic stress and depression onlaboratory rodents show long-term reduced concentration of neurosteroidsin the brain and in plasma, due to their reduced biosynthesis.

Similar findings are found in patients suffering from depression orpremenstrual syndrome. These findings point to, a violation ofhomeostatic mechanisms in the CNS of neuropsychiatric disorders relatedto stress.

Among well-known neurosteroids belong pregnenolone, progesterone,dehydroepiandrosterone (DHEA) and its reduced metabolite, and sulfateesters. The regulation of the synthesis of neurosteroids in the CNS isnot well known, but it is generally believed that the crutial isinteraction of various types of cells. For example, progesteronesynthesis by Schwann cells in peripheral nerves is regulated by diffusesignals from neurons.

Neurotrophic and neuroprotective effects of neurosteroids were shownboth in cell cultures and by in vivo experiments. Progesterone plays animportant role in neurological recovery from traumatic brain injury andspinal cord through mechanisms involving protection against excitotoxiccell damage, lipid peroxidation and induction of specific enzymes. Forexample, after spinal cord transection in rats progesterone increasesthe number of astrocytes expressing NO synthase just above and below thesite of transection.

Neurosteroids thus significantly modulate the function of membranereceptors for neurotransmitters, in particular the GABA_(A) receptor,NMDA receptor, and sigmal receptors. These mechanisms are responsiblefor psychopharmacological effects of steroids and partly explain theiranticonvulsant, anxiolytic, sedative and neuroprotective effects as wellas their influence on learning and memory processes.

For instance, pregnanolone sulfate was shown to be capable of reversingcognitive deficit in aged animals and exerting a protective effect onmemory in several amnesia models of amnesia. Current studies havedemonstrated direct effect of neurosteroids on intracellular receptors.Despite absence of direct evidence for binding of neurosteroids tocorticoid receptors, they may obviously modulate their functionindirectly, by interaction with protein kinases C and A, MAP-kinase orCaMKII. Moreover, pregnanolone and pregnanolone sulfate were shown toaffect microtubule-associated proteins and increase the rate ofmicrotubule polymeration, which may in turn affect neuronal plasticity.These newly described neurosteroid effects are still poorly understood,however, it can be assumed that they affect neuroprotectivity.

Sulfated and thus amphiphilic steroid compounds generally do notpenetrate the blood-brain barrier, but it was demonstrated thatintravenously administered pregnanolone sulfate reach the brain(Neuropharmacology 61, 61-68 (2011)). The transport of sulfated analogsis probably mediated by active exchange mechanisms associated withso-called organic anion transport protein (OATP), which is expressed inthe cells of brain tissue.

Inhibitors of the NMDA receptor are also some steroid derivatives and inparticular reduced derivatives of progesterone. Its neuroprotectiveproperties are also described in the patent literature (US2012/71453 A1,2012; WO 2009/108804 A and WO 2009/108809).

These drugs act only under spefic conditions of certain structuralprerequisites (J. Pharmacol. Exp. Ther. 293, 747-754 (2000)). Anessential structural requirement is bent shape of the molecule; thisrequirement is accomplished by derivatives 3alpha, 5beta-configurationof the steroid skeleton, and also to a lesser extent derivatives with3alpha, 5alpha-configuration. In addition, the activity is dependent onthe presence of ionisable groups within a convenient distance fromsteroid skeleton, i.e. 2 to 8 atoms. This group may be positively ornegatively charged. In previously published articles and patentliterature was also always mentioned as essential structural element ofthe acetyl substituent in position 17 of the steroid skeleton. Thisstructural element appears in progesterone, pregnenolone andpregnanolone (Br. J. Pharmacol. 166, 1069-1083 (2012); Steroids 76,1409-1418, (2011); WO 2009 108 804 and J. Med. Chem. 8, 426-432 (1965)).

The exception is the patent application U.S. Pat. No. 3,132,160 (1964patent was not granted) on derivatives of androstane with anesthetic andtranquility action. For the described compounds, however, ischaracterictic an oxygen atom at position C-11, analogously toclinically used pregnanolone analogue—alfaxalone. Due to the fact thatthe results of biological tests verifying biological properties of thesecompounds were never disclosed, we consider the activity specified inthis patent to be speculative.

Neuroprotective effect of steroid derivatives with a charged substituentat the C-3 also claimed by two patent applications: WO2010003391(Anionic pregnane compounds,method for Their Producing and Use of Them)and WO2012/110010 (Pregnanolone derivatives substituted in3alpha-position with the cationic group, Their method of production,usage and pharmaceutical preparation Involving Them). Both documentsclaim pregnane derivatives (polar substituent at C-20), substituted atC-3 of an anionic or cationic group of the formula

Derivatives claimed in the present application are not pregnanolonederivatives (these compounds do not have a keto group at the C-20). Incase that the claimed compound has a carbonyl group, for thestructure-activity reasons at position C-17 and thus, these compoundsare androstane derivatives. In case of other polar modification, thesederivatives have oxygen subtituent in a lower oxidation state (ether)than the C-20 keto group.

Removal of polar subtituent at position C-20, modifications andsubstitution of a D-ring at C-17 or C-16 with non-polar or lipophilicsubstituents, as well as complete removal of the steroidal D-ring, islikely to lead to better solubility of these derivatives in the membraneand a higher affinity to the NMDA receptor, resulting in some cases inmultiple reduction of IC₅₀ values in comparison with the referencecompound (3alpha, 5beta-pregnanolone sulfate). Our claimed compoundsshow that a higher degree of inhibition and IC₅₀ values lower than thereference compound can not generally be predicted, since thesubstitution or modification at the C- or D-ring in combination with thesize and composition of the substituent at C-3 is always unique and itis not possible to predict in advance and to suggest structure by anadditive approach. The above mentioned claims are illustrated byexamples of substances (Table II) with an IC₅₀ value lower than thereference compound.

DESCRIPTION OF THE INVENTION

The present invention relates to compounds with a protective effect onthe nervous system the structure of which comprises a substitutedtetradecahydrophenanthrene formula I.

They are useful in the treatment of traumatic brain injury, ischaemia,Alzheimer's and Parkinson's disease, inflammatory processes of thenervous system, vascular dementia, ischemia of fetuses and neonatals,neuropathic pain, or in similar processes in human and veterinarymedicine.

The invention also includes therapeutics composed from describedcompounds.

Tetradecahydrophenanthrene skeleton can have following configuration:

(2R,4aS,4bS,8aR,10aR)-tetradecahydrophenanthrene

(2S,4aS,4bS,8aR,10aR)-tetradecahydrophenanthrene

(2S,4aR,4bR,8aS,10aS)-tetradecahydrophenanthrene,

(4aS,4bS,8aR,10aR)-tetradecahydrophenantrene

The invention thus covers amphiphilic compounds withtetradecahydrophenanthrene skeleton of general formula I,

where

R¹ is selected from group of (—OSO₃pyH), (—OSO₃Na), (—OSO₃H),NaOOC-R⁶—C(R⁷)—R⁸—, HOOC—R⁶—C(R⁷)—R⁸—, HOOC—C(R⁷)—R⁸—, orR⁹R¹⁰—C(R¹¹)—R¹² —, where

-   -   R⁶ means straight or branched chain of C₁ to C₆ alkylene or C₂        to C₆ alkenylene, any of them unsubstituted or substituted with        one or more halogen atoms or amino group or amino group        protected by protecting groups, preferably by        tert-butylcarbonyl,    -   or R⁶ means trivalent —CH(CH₂—)₂ alkylene that forms with the        carbon carrying R⁷ and with R⁸ being nitrogen a five-membered        ring;    -   R⁷ means atom of oxygen, nitrogen or sulphur bound by double        bond or two atoms of hydrogen,    -   R⁸ means any at least divalent atom, preferably nitrogen, oxygen        or carbon,    -   R⁹ means a cationic group selected from groups of guanidinyl        formula,

-   -   -   or quaternary ammonium groups of the formula

-   -   -   where R¹³ to R²⁰ are hydrogen atoms or C₁ to C₆ alkyl or C₂            to C₆ alkenyl group with direct or branched chain,

    -   R¹⁰ means straight or branched C₁ to C₆ alkylene or C₂ to C₆,        both unsubstituted or substituted by one to 10 halogen atoms or        by amino group which is primary or substituted by C₁to C₄ alkyl        with direct or branched chain;

    -   R¹¹ is formed by atom of oxygen, nitrogen or sulphur bound by        double bond or by two atoms of hydrogen, and

    -   R¹² is chosen from group of oxygen, nitrogen or carbon and when        R¹² is carbon or nitrogen, its next valences are occupied by        hydrogen or hydrogens, while any of hydrogen can be substituted        by C₁ to C₄ alkyl or C₂ to C₄ alkenyl;

R² means hydrogen atom or methyl;

R³ means a) hydrogen atom and then

-   -   i) R⁴ and R⁵ are independently hydrogen atoms, or    -   ii) one of R⁴ and R⁵ means hydrogen atom and the other one means        straight or branched chain C₁ to C₆ alkyl or C₂ to C₆ alkenyl,        which is optionally substituted by 1 to 13 halogen atoms in case        of alkyl and by 1 to 9 halogen atoms in case of alkenyl, or by        atom of oxygen or sulphur bound by a double bond, while one of        the ethylene groups in the chain is optionally substituted by        oxygen or sulphur atom,    -   b) straight or branched C₁ to C₆ alkyl or C₂ to C₆ alkenyl,        which is optionally substituted by 1 to 13 halogen atoms in case        of alkyl or by 1 to 9 halogen atoms in case of alkenyl, or by        atom of oxygen or sulphur bound by a double bond, while one of        the methylene groups in the chain is optionally substituted by        oxygen or sulphur atom, and then R⁴ and R⁵ are independently to        each other hydrogen atoms, or    -   c) C₅ or C₆ alicyclic or aromatic substituent, while carbon        atoms can be functionalized by 1 to 8 atoms of halogen in case        of five-membered alicyclic ring, or 1 to 10 halogen atoms in        case of six-membered alicyclic ring or by 1 to 4 halogen atoms        in case of five-membered aromatic ring or 1-5 halogen atoms in        case of six-membered aromatic ring; and then        -   i) R⁵ is selected from group of hydrogen atom, or C₁ to C₆            alkyl or C₂ to C₆ alkenyl with direct or branched chain,            which is optionally substituted by 1 to 13 halogen atoms ift            case of alkyl and by 1 to 9 halogen atoms in case of            alkenyl, or by double bond bound atom of oxygen or sulphur,            while one of the methylene groups in the chain is optionally            substituted by oxygen or sulphur atom, or        -   ii) R⁴ and R⁵ means alkylene or alkenylene substituent            —(CH_(m))_(n)—, where n=3-4, m=1-2, forming with parent            carbon atoms of the skeleton at position 7 and 8 saturated            or unsaturated 5- or 6-membered cycle, where hydrogen atoms            of the aikenylen substituent are optionally substituted at            least by one halogen atom or C₁ to C₄ alkyl or C₂ to C₄            alkenyl, both with direct or branched chain; or one            methylene group of the alkylen substituent forming the ring            can be replaced by carbonyl group and the carbon atom at the            adjoining position can be substituted by another methylene            group, or it can be replaced by oxygen or sulphur atom,            while in case of sulphur atom, this can be further            functionalized by oxygen atom; or the hydrogens of one            methylene group of alkylen substituent can be replaced by            —O—CH₂—, to form an oxirane ring;    -   d) substituent —CH₂—O—CH(CH₃)—, then together with the first        carbon of alkylen group formed by R⁴ and R⁵, where R⁴ and R⁵        means alkylen substituent —(CH₂)₃— forms saturated and        methylated heretocycle;

and enantiomers of compound of general formula I, with the proviso thatin formula I are excluded compounds, where R¹ means HO₂C—R⁶CR⁷—R⁸—, R⁶means —(CH₂)₂—, R⁷ means oxygen atom bound by double bond and R⁸ meansoxygen atom, while R² and R³ mean methyls, R⁴ and R⁵ together formsgroup —(CH₂)₃— forming with parent carbon atoms oftetradecahydrophenanthrene skeleton at position 7 and 8 saturatedfive-membered ring; with absolute configuration 3R,5S,8S,9S,10S,13S,14S.

Halogen is chosen from group of —F, —Cl, —Br, —I.

Alkyl is straight or branched saturated hydrocarbon substituent, in oneembodiment containing one to six carbon atoms, in another embodimentfrom one to four carbon atoms, selected from methyl, ethyl, n-propyl,iso-propyl, n-butyl, sec-butyl, tert-butyl, isobutyl, amyl, t-amyl,isoamyl, n-pentyl, n-hexyl and similarly; removal of one hydrogen atomfrom the terminal alkyl CH₃ group will form corresponding alkylene.

Alkenyl is an unsaturated hydrocarbon substituent comprising dienes andtrienes of straight or branched chains containing 2 to 6 carbon atoms,or 2 to 4 carbon atoms, preferably selected from vinyl, allyl,1-butenyl, 2-butenyl, 1-pentenyl, 2-pentenyl, 3-pentenyl, 1-hexenyl,2-hexenyl, 3-hexenyl and similarly; removal of one hydrogen atom of theend group CH or CH₂ form the corresponding alkenylene.

Cycloalkyl or alicyclic group is selected from saturated cyclichydrocarbon radicals containing 3 to 6 carbon atoms, preferablycyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl.

The cycloalkenyl group is preferably selected from cyclopropenyl,1-cyclobutenyl, 2-cyclobutenyl, 1-cyclopentenyl, 2-cyclopentenyl,3-cyclopentenyl, 1-cyclohexenyl, 2-cyclohexenyl, 3-cyclohexenyl.

An aromatic group with six carbon atoms, either alone or in combinationwith other radicals, is preferably selected from phenyl.

Heterocycle or heterocyclic group is selected from C₃ to C₆ non-aromaticcyclic hydrocarbons containing one or more heteroatoms selected from O,N and S. Non-aromatic hydrocarbons containing the above heteroatoms, maybe saturated or partially saturated monocyclic radicals. AbbreviationpyH means pyridinium salt of particular sulfate.

In a preferred embodiment the present invention are the followingcompounds of Formula I:

pyridinium(2R,4aS,8aR,10aR)-4a-methyltetradecahydrophenanthren-2-yl2-sulfate (8),

pyridinium (2R,4aS, 4bS,7S,8S,8aS,10aR)-7-(methoxymethyl)-4a,7,8-trimethyltetradecahydrophenanthren-2-yl 2-sulfate(18),

4-(((2R,4aS,4bS,7S,8aS,10aR)-7-(methoxymethyl)-4a,7,8-trimethyltetradecahydrophenanthren-2-yl)oxy)-4-oxobutanoicacid (19),

pyridinium(2R,4aS,7S,8S,10aR)-7-(methoxycarbonyl)-4a,7,8-trimethyltetradecahydrophenanthren-2-yl2-sulfate(22),

4-(((2R,4aS,4bS,7R,8aS,10aR)-4a,7-dimethyltetradecahydrophenanthren-2-yl)oxy)-4-oxobutanoicacid (34),

pyridinium(2R,4aS,4bS,7R,8aS,10aR)-4a,7-dimethyltetradecahydrophenanthren-2-yl2-sulfate(35), methyl (2S,4aS,4bS,7R,8aR,10aS)-2,4b-dimethyl-7-(sulfooxy)tetradecahydrophenanthren-2-carboxylate(40),

pyridinium(3R,5R,8S,9S,10S,13S,14S)-10,13-dimethylhexadecahydro-1H-cyclopenta-[a]phenanthren-3-yl3-sulfate (49),

2-(((3R,5R, 8S,9S,10S,13S,14S)-10,13-dimethylhexadecahydro-1H-cyclopenta[a] phenanthren-3-yl)oxy)-2-oxoacetic acid (50),

2-(((3R,5R,8S,9S,10S,13S,14S)-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-3-yl)oxy)-2-oxopropanoicacid (51),

2-(((3R,5R,10S,13S,14S)-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-3-yl)amino)-2-oxoaceticacid (59),

((3R,5R,8S,9S,10S,13S,14S)-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-3-yl)amino)-3-oxopropanoicacid (61),

4-(((3R,5R,8S,9S,10S,13S,14S)-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-3-yl)oxy)-N,N,N-trimethyl-4-oxobutan-1-amonium chloride(62),

4-(((3R,5R,8R,9S,10S,13R,14S)-10,13-dimethyl-2,3,4,5,6,7,8,9,10,11,12,13,14,15-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl)oxy)-4-oxobutanoicacid (64),

3 -(((3R,5R,8R,9S,10S,13R,14S)-10,13-dimethyl-2,3,4,5,6,7,8,9,10,11,12,13,14,15-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl)oxy)-3-oxopropanoicacid (65),

3-(((3R,5R,8R,10S,13S,14S)-10,13-dimethyl-17-methylenehexadecahydro-1H-cyclopenta[a]phenanthren-3-yl)oxy)-3-oxopropanoicacid (67),

4-(((3R,5R,8R,9S,10S,13S,14S)-10,13-dimethyl-17-methylenhexadecahydro-1H-cyclopenta[a]phenanthren-3-yl)oxy)-4-oxobutanoicacid (68),

4-(((3R,5R,8R,9S,10S,13S,14S)-10,13-dimethyl-17-methylenhexadecahydro-1H-cyclopenta[a]phenanthren-3-yl)oxy)-4-oxopentanoicacid (69),

2-(((3R,5R,8R,9S,10S,13S,14S)-10,13-dimethyl-17-oxohexadecahydro-1H-cyclopenta[a]phenanthren-3-yl)aceticacid (74),

2-(((3R,5R,8R,9S,10S,13S,14S)-10,13-dimethyl-17-methylenhexadecahydro-1H-cyclopenta[a]phenanthren-3-yl)oxy)-N,N,N-trimethyl-2-oxoethan-1-ammoniumchloride (76),

3-(((3R,5R,8R,9S,10S,13S,14S,Z)-17-ethylidene-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-3-yl)oxy)-3-oxopropanoicacid (83),

5-(((3R,5R,8R,9S,10S,13S,14S,Z)-17-ethylidene-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-3-yl)oxy)-5-oxopentanoic acid (85),

3-(((3R,5R,8R,9S,10S,13S,14S,17R)-10,13-dimethyl-17-(prop-1-en-2-yphexadecahydro-1H-cyclopenta[a]phenanthren-3-yl)oxy)-3-oxopropanoicacid (88),

pyridinium (3R,5R,8R,9S,10S,13S,14S,17S)-17-iodo-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-3-yl3-Sulfate(93),

pyridinium(3R,5R,8R,9S,10S,13S,14S)-17,17-difluoro-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-3-yl3-Sulfate(95),

pyridinium (3R,5R,8R,9S,10S,13S,14S,17S)-10,13-dimethylhexadecahydrospiro[cyclopenta-[a]phenanthren-17,2′-oxiran]-3-yl3-Sulfate(97),

pyridinium(2R,4aS,4bS,6aS,10bS,6aS,12aR)-4a,6a-dimethyloctadecahydrochrysen-2-yl2-sulfate (101), (4S)-4-amino-5-(((2R,4aS,4bS,6aS,10bS,12aR)-4a,6a-dimethyloctadecahydrochtysen-2-yl)oxy)-5-oxopentanoicacid (106),

pyridinium(3R,5R,8S,9S,10S,13R,14S)-10,13-dimethyl-16-methylenhexadecahydro-1H-cyclopenta[a]phenanthren-3-yl3-sulfate(114),

pyridinium(3R,5R,8R,9S,10S,13S,14S)-10,13-dimethyl-17-methylenehexadecahydro-1H-cyclopenta[a]phenanthren-3-yl3-sulfate(116),

pyridinium(3R,5R,8S,9S,10S,13R,14S,17S)-10,13,17-trimethylhexadecahydro-1H-cyclopenta[a]phenanthren-3-yl3-sulfate (117),

pyridinium(3R,5R,8S,9S,10S,13S,14R,17R)-10,17-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-3-yl3-sulfate(118),

pyridinium(3R,5R,8S,9S,10S,13R,14R,17S)-10,17-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-3-yl3-sulfate (119),

pyridinium(3R,5R,8S,9S,10S,13R,14S,17S)-17-ethyl-10,13-dirnethylhexadecahydro-1H-cyclopenta[a]phenanthren-3-yl3-sulfate (120),

pyridinium(3R,5R,8R,9S,10S,13S,14S,17R)-10,13-dimethyl-17-(prop-1-en-2-yl)hexadecahydro-1H-cyclopenta[a]phenanthren-3-yl3-sulfate (121),

pyridinium(3R,5R,8R,9S,10S,13R,14S,17R)-17-isopropyl-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-3-yl3-sulfate (122),

pyridinium(3R,5R,8R,9S,10S,13R,14S,17R)-17-((R)-sec-butyl)-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-3-yl3-sulfate(123),

pyridinium(3S,3aS,5bR,7aR,9R,11aS,11bS,13aR)-3,11a-dimethylhexadecahydro-1H,3H-naphtho[2′,1′:4,5]indeno[1,7a-c]furan-9-yl9-sulfate(124),

pyridinium(3R,5R,8R,9R,10S,13S,14S)-13-methylhexadecahydro-1H-cyclopenta[a]phenanthren-3-yl3-sulfate(126),

pyridinium(3R,5S,8R,9R,10S,13S,14S)-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-3-yl3-sulfate (127),

pyridinium (2S,4aR,4bR,8aS,10aS)-4a-methyltetradecahydrophenanthren-2-yl2-sulfate (128),(4S)-4-amino-5-(((3R,5R,8S,9S,10S,13S,14S)-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-3-yl)oxy)-5-oxopentanoicacid (130),

1-((3R,5R,8S,9S,10S,13S,14S)-10,13-Dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-3-yl)-5-oxopyrrolidine-3-carboxylicacid (131), mixture of isomers sodium2-oxo-2-(((3R,5R,8S,9S,10S,13R,14S,17S)-10,13,17-trimethylhexadecahydro-1H-cyclopenta[a]phenanthren-3-yl)amino)acetate(138),

3-oxo-3-(((3R,5R,8S,9S,10S,13R,14S,17S)-10,13,17-trimethylhexadecahydro-1H-cyclopenta[a]phenanthren-3-yl)amino)propanoicacid (140),

sodium2-(((3R,5R,8R,9S,10S,13R,14S)-17-((R)-sec-butyl)-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-3-yl)amino)-2-oxoacetate(148),

3-(((3R,5R,8R,9S,10S,13R,14S)-17-((R)-sec-butyl)-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-3-yl)amino)-3-oxopropanoicacid (149),

2-(((3R,5R,8R,9S,10S,13 S,14S,Z)-17-ethylidene-10, I3-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-3-yl)amino)-2-oxoaceticacid (154),

3-(((3R,5R,8R,9S,10S,13S,14S,Z)-7-ethylidene-10,I3-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-3-yl)amino)-3-oxopropanoicacid (155).

Another object of the present invention are amphiphilic compounds withtetradecahydrophenanthrene skeleton of general formula I and thecorresponding specific compounds mentioned above, for use as amedicament.

Another aspect of the invention are also amphiphilic compounds withtetradecahydrophenanthrene skeleton of general formula I and thecorresponding above-mentioned specific compounds for use in treatingneuropsychiatric disorders associated with imbalances of glutamatergicneurotransmitter system, such as ischemic damage of the central nervoussystem, neurodegenerative changes and disorders of CNS, affectivedisorders, depression, post-traumatic stress disorder, and diseasesrelated to stress, anxiety, schizophrenia and psychotic disorders, pain,addiction, multiple sclerosis, epilepsy, glioma.

The present invention relates also to the use of amphiphilic compoundswith tetradecahydrophenanthrene skeleton of formula I for thepreparation of a veterinarian or human pharmaceutical drug orcomposition comprising it for treating of neuropsychiatric disordersassociated with imbalance of glutamatergic neurotransmitter system, suchas ischemic damage to the central nervous system, neurodegenerativechanges and disorders of CNS, affective disorders, depression,post-traumatic stress disorder, and diseases related to stress, anxiety,schizophrenia and psychotic disorders, pain, addiction, multiplesclerosis, epilepsy, glioma.

The invention further includes the use of amphiphilic compounds withtetradecahydrophenanthrene skeleton having the formula I for themanufacture of standard neuroprotective agents, antidepressants,antianxiety, mood stabilizers, hypnotives, sedatives, analgesics,anesthetics, antipsychotics, neuroleptics and procognitives oranalytical standards used in experimental research and analyticalchemistry or as compounds contained in food additives or cosmeticpreparations intended for improving the response of the individual partsof the organism to increased oxidative stress in particular, thenutrition and caused by free radicals, or by aging.

The invention also provides a pharmaceutical composition for human orveterinary use, comprising as active ingredient a compound having anamphiphilic tetradecahydrophenanthrene skeleton of general formula I orone of the above-mentioned specific amphiphilic compounds correspondingto general formula I.

Finally, the present invention also includes the above mentionedpharmaceutical composition for treating neuropsychiatric disordersassociated with an imbalance in glutamatergic neurotransmitter system,such as ischemic damage of CNS, neurodegenerative changes and disordersof CNS, affective disorders, depression, post-traumatic stress disorderand related diseases stress, anxiety, schizophrenia and psychoticdisorders, pain, addiction, multiple sclerosis, epilepsy, glioma.

The present invention will be further illustrated by Examples, whichshould not be construed as limiting the scope of the invention.

EXAMPLES ABBREVIATION LIST

-   CHCl₃ chloroform-   DMSO dimethylsulfoxide-   MS mass spectrometry-   HRMS high resolution mass spectrometry-   Boc tert-butoxycarbonyl-   EI electron ionization-   ESI electrospray ionization-   eq. eqvivalent-   IR infrared spectroscopy-   NMR nuclear magnetic resonance-   t-Bu tertial butyl-   Ac acetyl-   HEK human embryonic kidney cells-   GFP green fluorescent protein-   IC₅₀ the half maximal inhibitory concentration-   Opti-MEM® I minimum essential media, Invitrogen's product-   DHEA 5-dehydroepiandrosterone-   EGTA ethylene glycol tetraacetic acid-   EDTA ethylene diamine tetraacetic acid-   HEPES 4-(2-hydroxyethyl)-1-piperazineethanesuIfonic acid

Experimental Part—Chemistry

Melting points were measured at Hund Wetzlar H-600 (Helmut Hund,Germany). Samples for analysis were dried over phosphorous pentoxide at50° C. and a pressure of 100 mbar. Optical rotation was measured inchloroform Autopol IV polarimeter (Rudolph Research Analytical,Flanders, USA), [α]_(D) values are shown in 10⁻¹·deg·cm²·g and werecompensated to a standard temperature of 20° C. Infrared spectra weremeasured in chloroform or sample in potassium bromide tablets using aBruker IFS 55 wave numbers are given in cm⁻¹. ¹H NMR spectra weremeasured in FT mode at 24° C. and 400 MHz on a Bruker AVANCE-400 ordeuteromethanol or deuterochloroform with tetramethylsilane (TMS) asinternal standard. Chemical shifts are given in ppm (δ-scale), couplingconstants (J) are given in Hz. Signal multiplicities are designated asfollows: s—singlet, d—doublet, t—triplet, q—quartet, m—multiplet, bdenotes br (broad). All spectra were interpreted as the spectra of thefirst order. For describtion of NMR spectra, the classical cholesterolnumbering system was used. Mass spectra were measured on a ZAB-EQspectrometer (at 70 eV) or LCQ Classic (Thermo Finnigan). For thework-up, the aqueous hydrochloric acid solution (5%), or saturatedaqueous sodium bicarbonate were used. Thin layer chromatography (TLC)was performed on plates coated with a thin layer of silica gel (ICNBiochemicals). Preparative column chromatography was performed on silicagel Fluka (60 microns). For detection of the compounds on TLC plates wasused immersion in aqueous sulfuric acid solution (20 ml of 98% sulfuricacid) in methanol (250 ml) followed by heating at 300-400° C. Solventswere evaporated from the solution by rotary evaporation (0.25 kPa) at40° C. bath. The mobile phase for column chromatography is shown alwaysin the experiment. For the names of the compounds it is preferablyrecommended IUPAC nomenclature (PIN) and in cases where it was suitableterminology derived from appropriate steroid derivatives. For thepreparation of the active compounds tested were used de novo synthesisand modification of suitable commercially available precursors.

General Procedures General Procedure I—Synthesis of C-3 Sulfate

To a mixture of alcohol and sulphur trioxide -pyridine complex (2 eq.),dried under reduced pressure (30 min, 25° C., 100 Pa) was added freshlydried chloroform (10 ml per 100 mg) and dried pyridine (3 drops) and thereaction mixture was stirred under inert atmosphere at room temperaturefor 4 h. The reaction mixture was then cooled to −5° C. for 18 h, cooledand filtered through cotton wool. The filtrate was evaporated underreduced pressure and the residue is dried for 1 hour (25° C., 100 mbar).The residue was re-slurried in freshly dried dichloromethane (minimumvolume) and cooled to −5° C. for 2 h. The solids were filtered, thefiltrate evaporated under reduced pressure and dried (1 h, 25° C., 100mbar).

General Procedure II—Synthesis of C-3 Hemisuccinate

To a mixture of alcohol and succinic anhydride (7 eq., dried overnightat 50° C.) was added dry pyridine (5 ml per 100 mg) and4-(N,N-dimethylamino) pyridine (0.5 eq.). The reaction mixture washeated to 120° C. and the progress was monitored on TLC. The mixture wasthen poured into water and the product extracted with chloroform. Thecombined organic extracts were washed with saturated aqueous sodiumchloride solution and dried over anhydrous sodium sulfate. The solventswere evaporated under reduced pressure.

General Procedure III—Synthesis of C-3 Hemimalonate

Alcohol in dried toluene (5 mL per 100 mg) with pyridine (0.75 mL per100 mg) was added to a dry reaction flask with2,2-dimethyl-4,6-dioxo-1,3-dioxolane (Meldrum's acid, 1.1 eq.). Thereaction mixture was heated with stirring at 80° C. and the progress wasmonitored on TLC. It was then cooled to room temperature, diluted withwater and acidified with dilute hydrochloric acid (5%). The steroid wasextracted with ethyl acetate, the combined organic phases were washedwith dilute hydrochloric acid (5%), water and dried over anhydroussodium sulfate. The solvents were evaporated under reduced pressure.

General Procedure IV—Synthesis of C-3 Hemiglutarate

Alcohol and glutaric anhydride (258 mg, 2.26 mmol) were dried at 50° C.overnight. Then, dried pyridine (3 mL per 100 mg) and4-(N,N-dimethylamino)pyridine (0.3 eq.) were added. The mixture washeated at 120° C. and the progress was monitored on TLC. The reactionmixture was then cooled to room temperature, quenched by pouringreagents into water and the product extracted with chloroform. Thecombined organic extracts were washed with brine and dried withanhydrous magnesium sulfate. The solvents were evaporated under reducedpressure.

General Procedure V—Catalytic Hydrogenation

To a solution of the appropriate compound in ethanol (5 mL per 100 mg)and ethyl acetate (2.5 ml per 100 mg) was added the catalyst (Pd/CaCO₃,5%) and the mixture was vigorously stirred under a slight positivepressure of hydrogen at room temperature and the progress was monitoredon TLC. The catalyst was removed by filtration and the solventevaporated under reduced pressure.

General Procedure VI—Wilkinson Decarbonylation

Mixture of the appropriate compound andtris(triphenylphosphine)rhodium(I) chloride (1,1 eq.) in benzonitrile(24 ml per 1.3 g) was heated under inert atmosphere at 160° C. for 20 h. The reaction mixture was cooled to room temperature and filtered toremove a yellow solid. The filtrate was evaporated under reducedpressure.

General Procedure VII—Wittig Reaction Using n-Butyl Lithium

n-Butyl lithium (2.5M in hexane, 1.1 eq.) was added cold dropwise to asolution of methyltriphenylphosphonium iodide (1 eq.) in driedtetrahydrofuran (30 ml per 4 g) under an inert atmosphere of nitrogenand the mixture was stirred and heated at 80° C. for 2 hours. Then, asolution of compound (0.5 eq.) in dried tetrahydrofuran (minimum amount)was added. The reaction mixture was stirred at 80° C. and the progresswas monitored on TLC. The reaction was quenched with saturated ammoniumchloride solution. The product was extracted into chloroform; thecombined organic extracts were washed with brine and dried overanhydrous sodium sulfate. The solvents were evaporated under reducedpressure.

General Procedure VIII—Tosylation

A solution of particular compound, 4-dimethylaminopyridine (0.1 eq.),and p-TsCl (2 eq.) in anhydrous pyridine (75 ml per 4 g) was stirred atrt overnight. The reaction mixture was poured into icewater and theprecipitated white solid was collected by filtration, washed with waterand dried.

General Procedure IX—Substitution of Tosylate Protecting Group withAlkali Azide

A mixture of tosylated derivative and sodium azide (8 eq.) inN,N′-dimethylformamide (65 ml per 6 g) was heated under inert atmosphereat 55° C. for 4 h. Then, the reaction mixture was poured into water andthe product was extracted with ethyl acetate. The combined organicextracts were washed with 5% aqueous HCl, water, saturated aqueoussolution of sodium bicarbonate, water, dried and the solvents wereevaporated under reduced pressure.

General Procedure X—Reaction of C-3 Amino Group with Methyl3-Chloro-oxopropionate

A mixture of amine (202 mg, 0.52 mmol) in dry dichloromethane (4 ml per200 mg) was added dropwise to a cooled (0° C.) stirred mixture of methyl3-chloro-3-oxopropionate (3.5 eq.) and dry dichloromethane (3 ml per 0.2ml of the reagent) under inert atmosphere. The reaction mixture wasstirred at rt for 1.5 h, then it was poured into ammoniacal water,product was extracted with chloroform, the combined organic extractswere washed with brine, dried and the solvents were evaporated underreduced pressure.

General Procedure XI—Reaction of C-3 Amino Group with EthylChlorooxoacetate

A mixture of amine in dry benzene (15 ml per 100 mg) and pyridine (1 mlper 100 mg) was added dropwise to a cooled (0° C.) stirred mixture ofbenzene (10 ml per 100 mg) and ethyl chloro-oxoacetate (5 eq.) underinert atmosphere. The reaction mixture was stirred at room temperaturefor 1.5 h, The precipitated pyridine hydrochloride was filtered off,filtrate was washed twice with 5% aqueous sulfuric acid and then withwater. The solvent was evaporated under reduced pressure.

Example 1(S)-6-(Ethylendioxy)-8a-methyl-3,4,6,7,8,8a-bexahydronaphtalen-1(2H)-one(2)

Diketone 1 (7.36 g, 41.3 mmol), triethyl orthoformiate (7.58 ml, 45.6mmol), and ethylene glycol (12.7 ml, 228 mmol) were dissolved in DCM (50ml) and cooled to −10° C. Then, trifluoromethanesulfonate (150 ml, 830μmol) was added and the mixture was stirred at −10° C. for 1 h. Then,triethylamine (200 ml, 1.43 mmol) was added and the reaction mixture waspoured into saturated aqueous sodium bicarbonate solution. The productwas extracted into dichloromethane (3×50 ml). The combined organicextracts were dried over anhydrous magnesium sulfate and the solventsevaporated under reduced pressure. Residue was purified by columnchromatography on silica gel (150 g, 20% ethyl acetate in petroleumether) affording 7.88 g (86%) of monoketal 2: mp 48-51° C., [α]_(D)²⁰+97.7 (c 0.27, CHCl₃). ¹H NMR (400 MHz, CDCl₃): δ 1.32 (s, 3H, H-19),1.65 (qt, J₁=13.3, J₂=4.6, 1H, H-7b), 1.69-1.77 (m, 1H, H-1b), 1.77-1.89(m, 2H, H-2), 1.99-2.07 (m, 1H, H-7a), 2.16-2.08 (m, 1H, H-1b), 2.27(dddd, J₁=14.1, J₂=4.5, J₃=2.7, J₄=2.1, 1H, H-6b), 2.37 (dddd, J₁=15.2,J₂=4.7, J₃=2.9, J₄=1.8 Hz, 1H, CH-8b), 2.56 (dddd, J₁=14.0, J₂=13.5,J₃=5.0, J₄=1.9, 1H, H-6a), 2.64 (ddd, J₁=15.2, J₂=13.4, J₃=6.3 1H,H-8a), 5.41 (t, J=1.3, 1H, H-4), 3.87-4.02 (m, 4H, OCH₂CH₂O). ¹³C NMR(101 MHz, CDCl₃): δ 212.52 (C, C-9), 146.58 (C, C-5), 123.44 (CH, C-4),105.41 (C, C-3), 64.58 (CH₂, OCH₂CH₂O), 64.28 (CH₂, OCH₂CH₂O), 50.23 (C,C-10), 37.82 (CH₂, C-8), 30.82 (CH₂, C-6), 29.77 (CH₂, C-2), 28.60 (CH₂,C-1), 24.27 (CH₂, C-7), 23.81 (CH₃, C-19). IR spectrum (CHCl₃): 2953,1461, 1447, 1442 (CH₂); 2975 2885 (CH₃); 1711 (C═O); 1661 (C═C). MS(ESI) m/z: 223 (40%, (M+H), 245 (100%, M+Na). HR-MS (ESI) m/z: ForC₁₃H₁₈NaO₃ (M+Na) calcd 245.1148; found: 245.1148. For C₁₃H₁₈O₃ (222.3)calcd: 70.24% C, 8.16% H; found: 69.86% C, 8.19% H.

Example 2(S)-7-(Ethylenedioxy)-4b-methyl-1,2,4b,5,6,7,9,10-oetabydrophenanthren-3(4H)-one(3a) and(4bS,10aS)-7-(ethylenedioxy)-4b-methyl-1,2,4b,5,6,7,10,10a-octahydrophenanthren-3(9H)-one(3b)

Sodium hydride (9.39 g, 235 mmol, 60% suspension in oil, washed withtetrahydrofuran, 3×25 ml) in tetrahydrofuran (10 ml) was added to acooled (0° C.) solution of ketone 2 (20.87 g, 93.89 mmol) in dry ethylformate (250 ml). Then, methanol (3.80 ml, 93.9 mmol) was added dropwiseover 15 min at 0° C. The reaction mixture got thickened within a fewminutes and after 30 minutes was warmed to room temperature. After anadditional 30 min, it was quenched with saturated ammonium chloridesolution (400 ml) and the product was extracted with ethyl acetate(3×100 ml). The combined organic extracts were dried over anhydroussodium sulfate, the solvent evaporated under reduced pressure to afforda quantitative amount of the crystalline formyl derivative: ¹H NMR (400MHz, CDCl₃): δ 1.37 (3H, s, CH₃-19), 1.82-2.13 (4H, m, CH₂-1, CH₂-2),2.22-2.43 (2H, m, CH₂-6), 2.30-2.57 (2H, m, CH₂-7), 3.84-4.05 (4H, m,OCH₂CH₂O), 5.39 (1H, t, J=1.1, CH-4), 8.54 (1H, s, CHOH), 14.67 (1H, bs,OH). ¹³C NMR (101 MHz, CDCl₃): δ 190.20 (C, C-9), 185.76 (CH, CHOH),144.96 (C, C-5), 122.52 (CH, C-4), 106.76 (C, C-8), 105.32 (C, C-3),64.72 (CH₂, OCH₂CH₂O), 64.26 (CH₂, OCH₂CH₂O), 41.98 (C, C-10), 30.34(CH₂, C-1), 29.89 (CH₂, C-2), 29.14 (CH₂, C-6), 24.28 (CH₂, C-7), 23.75(CH₃, C-19).

Butenone (8.77 ml, 105 mmol) and triethylamine (245 ml, 1.75 mmol) wasadded to the formyl derivative and the reaction mixture was stirredovernight. Excess of butenone was evaporated under reduced pressure andthe crude mixture was dissolved in methanol (320 ml) solution was addedto an aqueous solution of potassium hydroxide (15.26 g, 272 mmol) andthe mixture was heated to reflux under inert atmosphere for 30 min. Thesolution was cooled to room temperature, quenched with saturatedammonium chloride solution (400 ml) and the product was extracted withethyl acetate (3×330 ml). The combined organic extracts were washed withsaturated aqueous sodium chloride (400 ml) and dried over anhydroussodium sulfate. The solvents were evaporated under reduced pressure andthe residue chromatographed on silica gel (350 g, 0.5% triethylamine and20%-50% ethyl acetate in petroleum ether) affording 5.49 g (22%) of thederivative 3b and 14.53 g (5 8%) of the derivative 3a.

Compound 3a: mp 105-108° C., [α]_(D) ²⁰+149.8 (c 0.26, CHCl₃). ¹H NMR(400 MHz, CDCl₃): δ 1.31 (3H, s, CH₃-19), 1.67 (1H, dtd, J₁=14.1,J₂=12.8, J₃=4.6, CH-7b), 1.61 (1H, ddd, J₁=13.8, J₂=10.2, J₃=4.6,CH-14b), 1.91-1.82 (1H, m, CH-1b), 1.85-1.94 (2H, m, CH₂-2), 1.93-2.00(1H, m, CH-7a), 2.00-2.10 (1H, m, CH-1a), 2.05-2.14 (1H, m, CH-14a),2.21 (1H, ddd, J₁=14.0, J₂=3.9, J₃=2.8, CH-6b), 2.30 (1H, ddd, J₁=16.5,J₂=14.1, J₃=5.0, CH-13b), 2.39-2.45 (1H, m, CH-13a), 2.47 (1H, tdd,J₁=14.0, J₂=4.4, J₃=1.9, CH-6a), 2.68 (1H, ddtd, J₁=12.3, J₂=10.1 ,J₃=5.0, J₄=2.2, CH-8), 3.86-4.06 (4H, m, OCH₂CH₂O), 5.36 (1H, d, J=1.3,CH-4), 5.97 (1H, d, J=2.1, CH-11). ¹³C NMR (101 MHz, CDCl₃): δ 200.34(C, C-12), 172.37 (C, C-9), 147.21 (C, C-5), 122.65 (CH, C-11), 122.26(CH, C-4), 105.40 (C, C-3), 64.67 (CH, OCH₂CH₂O), 64.33 (CH, OCH₂CH₂O),41.39 (C, C-10), 36.42 (CH₂, C-13), 35.22 (CH, C-8), 34.11 (CH₂, C-7),32.06 (CH₂, C-1), 31.43 (CH₂, C-6), 30.02 (CH₂, C-2), 29.51 (CH₂, C-14),27.20 (CH₂, C-19). IR spectrum (CHCl₃): 2978 (CH₃); 2941 (CH₂); 2887(CH₃); 2865 (CH₂); 1664 (C═O); 1604 (C═C); 1454, 1451 (CH₂); 1379 (CH₃);1361, 1168, 1132 (CH₂); 1091, 1078, 946, 883 (kruh). MS (ESI) m/z: 275(57%, M+H), 297 (100%, M+Na). HR-MS (ESI) m/z: For C₁₇H₂₃O₃ (M+H) calcd:275.1642; found: 275.1644. For C₁₇H₂₂O₃(274.2) calcd: 74.42% C, 8.08% H;found: 74.29% C, 7.98% H. Oily product 3b: [α]_(D) ²⁰+217.7 (c 0.22,CHCl₃). ¹H NMR (400 MHz, CDCl₃): δ 1.18 (3H, s, CH₃-19), 1.60-1.78 (2H,m, CH₂-7), 1.74-1.98 (2H, m, CH₂-6), 2.12-2.23 (1H, m, CH-2b), 2.29-2.36(2H, m, CH₂-14), 2.34-2,44 (1H, m, CH-2a), 2.33-2.46 (2H, m, CH₂-13),2.76 (1H, bd, J₁=20.1, J₂=1.1, CH-11b), 2.89 (1H, bd, J₁=20.1, J₂=1.6,CH-11a), 3.81-4.08 (4H, m, OCH₂CH₂O), 5.33 (1H, t, J=1.6, CH-4). ¹³C NMR(101 MHz, CDCl₃): δ 211.2 (C, C-12), 148.44 (C, C-5), 132.39 (C, C-9),128.58 (C, C-8), 120.22 (CH, C-4), 105.62 (C, C-3), 64.67 (CH,OCH₂CH₂O), 64.17 (CH, OCH₂CH₂O), 38.50 (CH₂, C-11), 38.04 (CH₂, C-13),37.63 (C, C-10), 32.44 (CH₂, C-7), 32.26 (CH₂, C-1), 30.17 (CH₂,C-6/C-14), 30.09 (CH₂, C-6/C-14), 29.09 (CH₂, C-2), 23.02 (CH₃, C-19).IR spectrum (CHCl₃): 2954, 2927 (CH₂); 2855 (CH₂); 1713 (C═O); 1674(C═C); 1450, 1443 (CH₂); 1380 (CH₃); 1363, 1137 (CH₂); 1086, 946, 961(ring). MS (ESI) m/z: 275 (100%, M+H), 297 (42%, M+Na). (ESI) m/z: ForC₁₇H₂₃O₃ (M+H) calcd: 275.1642; found: 275.1644. For C₁₇H₂₂O₃ (274.4)calcd: 74.42% C, 8.08% H; found: 74.80% C, 8.82% H.

Example 3(4bS,10aS)-7-(Ethylendioxy)-4b-methyl-1,2,4,4a,4b,5,6,7,10,10a-decahydrophenanthren-3(9H)-one(4)

To the dried (lithium wire and a catalytic amount of ferric chloride)freshly distilled liquid ammonia in a three-necked flask, cooled to −78°C. under a condenser with solid carbon dioxide was under a nitrogenatmosphere, a solution of enone 3a (9.074 g, 33.07 mmol) intetrahydrofuran (90 ml) was added followed by ethanol (4.96 ml, 84.9mmol). Then, under intensive stirring, lithium metal (2.66 g, 383 mmol)cut in small pieces were added portionwise. When a persistent bluecoloration showed complete reduction, excess ammonia was gentlyevaporated. The residue was poured into saturated aqueous sodiumbicarbonate (300 ml) and the product was extracted into ethyl acetate(3×100 ml). The combined organic extracts were washed with saturatedaqueous sodium chloride solution, dried over anhydrous magnesium sulfateand evaporated under reduced pressure. The residue was purified bycolumn chromatography on silica gel (150 g, 0.5% triethylamine, 30%ethyl acetate in petroleum ether) to afford 7.63 g (83%) of 4 as acolorless oil: [α]_(D) ²⁰+144.7 (c 0.41, CHCl₃). ¹ NMR (400 MHz, CDCI₃):δ 1.07 (3H, s, CH₃-19), 1.10-1.17 (1H, m, CH-7b), 1.25-1.34 (1H, m,CH-14b), 1.30-1.47 (1H, m, CH-9), 1.51-1.61 (1H, m, CH-1b), 1.64-1.69(1H, m, CH-1a), 1.72-1.82 (2H, m, CH₂-2), 1.75-1.85 (1H, m, CH-8),1.81-1.89 (1H, m, CH-7a), 1.97-2.04 (1H, m, CH-14a), 2.07-2.14 (1H, m,CH-6b), 2.09-2.17 (1H, m, CH-11 b), 2.24-2.32 (1H, m, CH-6a), 2.28-2.38(2H, m, CH₂-13), 2.33-2.44 (1H, m, CH-11a), 3.82-4.07 (4H, m, OCH₂CH₂O),5.31 (1H, t, J=1.2, CH-4). ¹³C NMR (101 MHz, CDCl₃): δ 211.82 (C, C-21),149.20 (C, C-5), 121.12 (CH, C-4), 105.77 (C, C-3), 64.61 (CH,OCH₂CH₂O), 64.25 (CH, OCH₂CH₂O), 53.03 (CH, C-9), 41.09 (CH₂, C-11),40.88 (CH₂, C-13), 37.65 (C, C-10), 35.88 (CH, C-8), 34.09 (CH₂, C-1),33.59 (CH₂, C-7), 33.34 (CH₂, C-14), 31.57 (CH₂, C-6), 29.66 (CH₂, C-2),17.55 (CH₃, C-19). IR spectrum (CHCl₃): 2969 (CH₃); 2938 (CH₂); 2888,2864 (CH₃); 1711 (C═O); 1664 (C═C); 1440 (CH₂); 1381, 1366 (CH₃); 1089,1233, 1182, 1169, 1113 (COCOC); 1009, 964, 947 (ring). MS (ESI) m/z: 277(23%, M+H), 299 (100%, M+Na), 575 (21%, 2M+Na). HR-MS (ESI) m/z: ForC₁₇H₂₅O₃ (M+H) calcd: 277.17982; found: 277.17993. For C₁₇H₂₄NaO₃ (M+Na)calcd: 299.16181. For C₁₇H₂₄O₃ (276.4) calcd: 73.88% C, 8.75% H; found:74.01% C, 8.69, % H.

Example 4(4aS,4bR,10aR)-7,7-Dimethoxy-4b-methyl-1,2,3,4,4a,4b,5,6,7,9,10,10a-dodecahydro-phenanthrene(5)

To a stirred solution of the ketone 4 (1.00 g, 3.62 mmol) in methanol(25 ml) at room temperature was added tosylhydrazide (1.01 g, 5.42mmol). After 30 minutes, sodium borohydride (2.74 g, 72.4 mmol) wasadded over 1 h while stirring and cooling to 25° C. The reaction mixturewas stirred overnight and then poured into water (100 ml) and theproduct extracted with n-pentane (3×20 ml). The combined organicextracts were washed with saturated aqueous sodium chloride solution,dried over anhydrous magnesium sulfate and the solvents evaporated underreduced pressure. Chromatography on silica gel (30 g, 3% ethyl acetatein n-pentane) afforded 708 mg (75%) of the ketal 5: [α]_(D) ²⁰+123.2 (c0.57, CHCl₃). ¹H NMR (400 MHz, CDCl₃): δ 0.83-0.94 (1H, m, CH-9),0.87-0.97 (1H, m, CH-12b), 0.99 (3H, s, CH₃-19), 0.95-1.04 (1H, m,CH-14b), 1.01-1.15 (1H, m, CH-7b), 1.12-1.23 (2H, m, CH-11b, CH-13b),1.35 (1H, qt, J₁=11.2, J₂=3.6, CH-8), 1.52-1.61 (1H, m, CH-1b),1.61-1.70 (3H, m, CH-7a, CH-11a, CH-12a), 1.65-1.75 (1H, m, CH-14a),1.70-1.80 (4H, m, CH-1a, CH₂-2, CH-13a), 2.02 (1H, ddd, J₁=13.7, J₂=4.3,J₃=2.4, CH-6b), 2.22 (1H, tdd, J₁=13.7, J₂=4.7, J₃=1.6, CH-6a),3.85-4.04 (4H, in, OCH₂CH₂O), 5.24 (1H, d, J=1.4, CH-4). ¹³C NMR (101MHz, CDCI₃): δ 151.86 (C, C-5), 119.58 (CH, C-4), 106.29 (c, C-3), 64.54(CH₂, OCH₂CH₂O), 64.20 (CH₂, OCH₂CH₂O), 53.47 (CH, C-9), 37.56 (C,C-10), 37.11 (CH, C-8), 35.22 (CH₂, C-7), 34.68 (CH₂, C-12), 34.56 (CH₂,C-1), 32.14 (CH₂, C-6), 29.93 (CH₂, C-2), 26.80 (CH₂, C-13), 26.25 (CH₂,C-11), 25.61 (CH₂, C-14), 17.92 (CH₃, C-19). IR spectrum (CHCl₃): 2970(CH₃); 2927 (CH₂); 2886, 2855 (CH₃); 1659 (C═C); 1451 (CH₂); 1451 (CH₂);1380, 1364 (CH₃); 1233 (COCOC); 1170, 1113 (ketal); 1086, 1014, 954, 947(C—O—C). MS (ESI) m/z: 263 (23%, M+H), 285 (13%, M+Na). HR-MS (ESI) m/z:For C₁₇H₂₆NaO₂ (M+Na) calcd: 285.18250; found: 285.18243. For C₁₇H₂₆O₂(262.4) calcd: 77.82% C; 9.99% H; found: 77.94% C, 10.08% H.

Example 5 (4aS,4bS,8aR,10aR)-4a-Methyldodecahydrophenanthren-2(1H)-one(6)

To a solution of the ketal 5 (380 mg, 1.45 mmol) in acetone (10 ml) andwater (0.5 ml) was added hydrochloric acid (35%, 3 drops) and thereaction mixture was stirred at room temperature overnight. Then, thesolution was concentrated on a rotary evaporator, poured into aqueoushydrochloric acid (5%, 30 ml) and the product extracted with n-pentane(3×20 ml). The combined organic extracts were washed with saturatedaqueous sodium bicarbonate solution, dried over anhydrous sodium sulfateand the solvents evaporated under reduced pressure. The residue wasdissolved in ethanol (20 ml) and potassium hydroxide (45 mg) in water(120 ml) and catalyst (Pd/CaCO₃, 5%, 40 mg) were added. The mixture washydrogenated under a slight positive pressure of hydrogen for 3 h. Thecatalyst was then filtered off, the solvent partly evaporated and theresidue poured into water. The product was extracted with n-pentane(3×20 ml). The combined organic extracts were dried over anhydrousmagnesium sulfate and evaporated in vacuo rotary evaporator afforded 291mg (91%) of ketone 6, the product consisted of a mixture of isomers 5αand 5β 1:9. Ketone 6: [α]_(D) ²⁰+27.8 (c 0.43, CHCl₃). ¹H NMR (400 MHz,CDCl₃) δ 0.97 (3H, s, CH₃-19), 0.98-1.07 (1H, m), 1.15-1.27 (2H, m,CH-6b, CH-7b), 1.31-1.41 (1H, m, CH-1b), 1.42-1.49 (2H, m, CH-7a, CH-9),1.65-1.72 (4H, m), 1.77-1.86 (2H, m, CH-5), 1.85-1.96 (1H, m, CH-6a),1.98-2.06 (1H, ddd, J₁=14.9, J₂=4.7, J₃=2.4, CH-4b), 2.02-2.10 (1H, m,CH-2b), 2.16 (1H, dddd, J₁=14.7, J₂=4.3, J₃=2.5, CH-1a), 2.37 (1H, tdd,J₁=14.7, J₂=5.5, J₃=0.9, CH-2a), 2.72 (1H, dd, J₁=14.9, J₂=13.3, CH-4a).¹³C NMR (101 MHz, CDCl₃): δ 213.39 (C, C-3), 44.53 (CH, C-5), 42.34(CH₂, C-4), 40.36 (CH, C-9), 37.29 (CH₂, C-4), 36.92 (CH, C-8), 36.46(CH₂, C-1), 35.10 (CH₂), 35.00 (C, C-10), 28.42 (CH₂, C-7), 27.26 (CH₂),26.51 (CH₂), 26.45 (CH₂), 25.61 (CH₂), 22.69 (CH₃, C-19). IR spectrum(CHCl₃): 2981, 2929, 2856 (CH₂); 1707 (C═O); 1455, 1448 (CH₂); 1382(CH₃). MS (EI) m/z: 149 (100%, (M−C₄H₅O), 220 (66%, M). HR-MS (ESI) m/z:For C₁₅H₂₄O (M⁺) calcd: 220.1822; found: 220.1825. For C₁₅H₂₄O (220.4)calcd: 81.76% C; 10.98% H; found: 81.61% C; 11.03% H.

Example 6 (2R,4aS,4bS,8aR,10aR)-4a-Methyltetradecahydrophenanthren-2-ol(7a)

A mixture of ketone 6 (274 mg, 1.24 mmol), dichloromethane (5 ml) anddry methanol (5 ml) was cooled to −78° C. Then, dried cerium chloride(337 mg, 1.37 mmol) and sodium borohydride (52 mg, 1.37 mmol) were addedwhile stirring. After 15 min stirring at −78° C. the reaction mixturewas slowly warmed to room temperature and quenched with dilutehydrochloric acid (5%, 25 m1). The product was extracted withdichloromethane (3×10 ml), the combined organic extracts were washedwith saturated aqueous sodium bicarbonate solution and dried overanhydrous magnesium sulfate. Evaporation of the solvents andchromatography of the residue on silica gel column (10 g, 10% ether inn-pentane) yielded 186.6 mg (68%) of 3α, 5β-7a alcohol and 5.5 mg (2%),3β,5α-alcohol 7b. Compound 7a: [α]_(D) ²⁰+21.8 (c 0.29, CHCl₃). ¹H NMR(400 MHz, CDCl₃): δ 0.87 (3H, s, CH₃-19), 0.84-0.96 (1H, m, CH-7b),0.90-1.04 (2H, m, CH-1b, CH-14b), 1.14-1.24 (2H, m, CH-13b, CH-11b),1.15-1.29 (2H, m, CH-6b, CH-12b), 1.24-1.33 (1H, m, CH-8), 1.32-1.39(1H, m, CH-12a), 1.35-1.42 (1H, m, CH-5), 1.31-1.44 (1H, m, CH-2b),1.39-1.46 (1H, m, CH-9), 1.50 (1H, dddd, J₁=12.6, J₂=4.7, J₃=3.8,J₄=2.4, CH-4b), 1.58-1.66 (2H, m, CH-1a, CH-7a), 1.60-1.70 (1H, m,CH-11a), 1.62-1.70 (1H, m, CH-2a), 1.71-1.80 (1H, m, CH-13a), 1.72-1.84(1H, m, CH-4a), 1.77-1.87 (1H, m, CH-14a), 1.84-1.91 (1H, m, CH-6a),2.18 (1H, bs, OH), 3.62 (1H, tt, J₁=11.1, J₂=4.7, CH-3). ¹³C NMR (101MHz, CDCl₃): δ 71.73 (CH, C-3), 42.22 (CH, C-5), 40.00 (CH, C-9), 37.25(CH, C-8), 36.21 (CH₂, C-4), 35.17 (CH₂, C-1), 34.64 (CH₂, C-14), 34.53(C, C-10), 30.54 (CH₂, C-2), 29.09 (CH₂, C-12), 27.28 (CH₂, C-13), 27.02(CH₂, C-6), 26.53 (CH₂, C-11), 25.34 (CH₂, C-7), 23.34 (CH₃, C-19). IRspectrum (CHCl₃): 3609, 3452 (OH); 2977 (CH₂); 2927 (CH₂); 2858 (CH₂);1450 (CH₂); 1380, 1364 (CH₃); 1035, 1015 (C—OH). MS (ESI) m/z: 245(100%, M+Na). HR-MS (ESI) m/z: For C₁₅H₂₆NaO (M+Na) calcd: 245.1876;found: 245.1875. For C₁₅H₂₆O (222.4) calcd: 81.07% C, 11.79% H; found:81.11% C, 11.98% H.

Example 7 Pyridinium(2R,4aS,4bS,8aR,10aR)-4a-methyltetradecahydrophenanthren-2-yl 2-Sulfate(8)

Compound 8 was prepared according to General Procedure I—Preparation ofC-3 Sulfate from compound 7a (166 mg , 747 μmol) affording sulfate 8(248 mg, 87%): [α]_(D) ²⁰+22.6 (c 0.23, CHCl₃). ¹H NMR (400 MHz, CDCl₃):δ 0.86 (3H, s, CH₃-19), 0.83-0.93 (1H, m, CH-11b), 0.90-1.00 (1H, m),0.95-1.04 (1H, m, CH-1b), 1.22-1.35 (1H, m, CH-8), 1.12-1.40 (5H, m,CH₂-6), 1.38-1.48 (2H, m, CH-5, CH-9), 1.54-1.63 (1H, m, CH-11a),1.50-1.64 (1H, m, CH-2b), 1.56-1.66 (2H, m), 1.71-1.78 (1H, m),1.79-1.88 (3H, m, CH-1a, CH-4b), 1.90-2.03 (2H, m, CH-4a, CH-2a), 4.47(1H, tt, J₁=11.3, J₂=4.9, CH-3), 7.99-8.03 (2H, m, CH-3′), 8.48 (1H, tt,J₁=7.9, J₂=1.6, CH-4′), 8.99-8.01 (2H, m, CH-2′). ¹³C NMR (101 MHz,CDCl₃): δ 145.60 (CH, C-4′), 142.37 (CH, C-2′), 127.12 (CH, C-3′), 79.67(CH, C-3), 42.34 (CH, CH-5), 40.05 (CH, C-9), 37.29 (CH, C-8), 35.29(CH₂), 34.67 (CH₂, C-1), 34.52 (C, C-10), 33.26 (CH₂, C-4), 29.09 (CH₂,C-6), 27.89 (CH₂, C-2), 27.38 (CH₂), 26.93 (CH₂), 26.61 (CH₂), 25.41(CH₂, C-11), 23.32 (CH₃, C-19). IR spectrum (CHCl₃): 2927 (CH₂); 2856(CH₂); 2450-2750 (NH⁺); 2135 (NH⁺); 1490 (ring); 1450 (CH₂); 1380 (CH₃);1255, 1171, (SO₃); 1047 (SO₃); 970, 953, (COS); 828 (COS); 682 (═CH);624 (SO₃). MS (ESI) m/z: 301 (100%, M-C₅H₆N⁺). HR-MS (ESI) m/z: ForC₁₅H₂₅O₄S (M-C₅H₆N⁺) calcd: 301.1479; found: 301.1479. For C₂₀H₃₁NO₄S(381.5) calcd: 62.96% C, 8.19% H; 3.67% N; found: 60.82; % C, 8.09% H;3.61% N.

Example 84-(((2R,4aS,4bS,8aR,10aR)-4a-Methyltetradecahydrophenanthren-2-ypoxy)-4-oxobutanoicAcid (9)

Compound 9 was prepared according to General Procedure 11—Preparation ofC-3 Hemisuccinate from compound 7a (47 mg, 0.21 mmol). Chromatography onsilica gel (4-10% acetone in petroleum ether) afforded 52 mg (76%) ofcompound 9: mp 131-133° C. (acetone/n-heptane), [α]_(D) ²⁰+45.5 (c 0.20,CHCl₃). ¹H NMR (400 MHz, CDCl₃): δ 0.87 (3H, s, H-19), 2.51-2.76 (4H, m,H-2′ and H-4′), 4.76 (1H, tt, J₁=11.3, J₂=4.7, H-3). ¹³C NMR (101 MHz,CDCl₃): δ 177.10, 171.83, 75.31, 42.31, 40.26, 37.47, 35.38, 34.84,34.55, 32.30, 29.45, 29.24, 29.04, 27.49, 27.10, 26.89, 26.75, 25.59,23.53. IR spectrum (CHCl₃): 3517 (OH, monomer); 1727, 1717 (C═O); 1232,1176, 1170 (COC). MS (ESI) m/z: 345.2 (100%, M+Na). HR-MS (ESI) m/z: ForC₁₉H₃₀O₄Na (M+Na) calcd: 345.2036; found: 345.2036.

Example 9 Methyl(1S,2S,4aS,4bS,7R,8aR,10aR)-7-acetoxy-2,4b-dimethyl-1-(2-oxoethyl)tetradeca-hydrophenanthren-2-earboxylate(11)

A stirred solution of enol acetate 10 (5.0 g , 13.35 mmol) indichloromethane (150 ml) and glacial acetic acid (13 ml) was ozonized at—78° C. until a blue color of the solution persisted. Then, to thereaction mixture was gradually added in small portions dimethylsulfide(2 ml, 27.37 mmol), glacial acetic acid (130 ml) and water (28 ml). Theresulting solution was stirred 18 hours at room temperature. The productwas extracted into dichloromethane, the combined organic extracts werewashed with water, dried over anhydrous magnesium sulfate and thesolvents were evaporated under reduced pressure on a rotary evaporator.The residue was dissolved in ether, the solution cooled to 0° C. andaddition of an ethereal solution of diazomethane was added and freecarboxylic group esterified. Chromatography on silica gel (10% ethylacetate in petroleum ether) afforded 4.45 g (88%) non-crystallisingmethyl ester 11: [α]_(D) ²⁰-9.6 (c 0.24, MeOH). ¹H NMR (400 MHz, CD₃OD):δ 0.89 (3H, s, H-18), 1.11 (3H, s, H-19), 2.02 (3H, s, OAc), 3.65 (3H,s, OCH₃), 4.66-4.76 (1H, m, H-3), 9.67 (1H, s, CHO). ¹³C NMR (101 MHz,CD₃OD): δ 201.97, 178.37, 170.76, 74.08, 52.12, 47.59, 46.71, 41.69,41.46, 39.83, 37.80, 36.78, 34.88, 34.77, 32.10, 26.96, 26.72, 26.31,23.33, 21.55, 19.83, 15.56. IR spectrum (CHCl₃): 2828 (CHO); 1721 (C═O);1435, 1385, 1364 (CH₃); 1253 (COO). MS (ESI) m/z: 401.3 (100%, M+Na).HR-MS (ESI) m/z: For C₂₂H₃₄O₅Na (M+Na) calcd: 401.2299; found: 401.2297.

Example 10 Methyl(1S,2S,4aS,4bS,7R,8aR,10aS)-7-acetoxy-1,2,4b-trimethyltetradecabydro-phenanthren-2-earboxylate(12)

Compound 12 was prepared according to General Procedure VI—WilkinsonDecarbonylation from compound 11 (1.33 g, 3.65 mmol). Chromatography onsilica gel (10% acetone in petroleum ether) afforded 1.31 g (69%) of 12:¹H NMR (400 MHz, CD₃OD): 6 0.72 (311, d, J=6.7, H-15), 0.89 (31-1, s,H-18), 1.06 (3H, s, H-19), 2.03 (31⁻1, s, OAc), 3.66 (3H, s, OCH₃),4.66-4.76 (1H, m, H-3). ¹³C NMR (101 MHz, CD₃OD): δ 179.21, 170.77,74.35, 51.85, 47.76, 42.36, 41.56, 39.66, 37.74, 37.05, 34.86 (2×C),32.19, 27.07, 26.75, 25.52, 23.36, 21.58, 20.09, 15.34, 14.64. IRspectrum (CHCl₃): 1721 (C═O); 1467, 1386, 1024 (OAc); 1364, 1160(COOCH₃). MS (ESI) m/z: 373.2 (100%, M+Na). HR-MS (ESI) m/z: ForC₂₁H₃₄O₄Na (M+Na) calcd: 373.2349; found: 373.2348.

Example 11(2R,4aS,4bS,7S,8S,8aS,10aR)-7-(Hydroxymethyl)-4a,7,8-trimethyltetradecahydro-phenanthren-2-ol(13)

A mixture of ester 12 (1.00 g, 2.86 mmol) and lithium aluminum hydride(2.86 mg, 8.58 mmol) in tetrahydrofuran (30 ml) was heated to refluxunder an inert atmosphere of argon for 2 h. The excess of reagent wascarefully quenched with saturated aqueous sodium sulfate; inorganicmaterials were removed by filtration and washed with ethyl acetate. Thefiltrate was washed with aqueous hydrochloric acid (5%), water,saturated aqueous sodium bicarbonate solution and dried over anhydroussodium sulfate. The solvents were evaporated under reduced pressure. Theattemps to crystallize the residue 585 mg (73%) 13 failed: [α]_(D)²⁰−3.4 (c 0.33, CHCl₃). ¹H NMR (400 MHz, CD₃OD): δ 0.69 (3H, s, H-18),0.78 (3H, d, J, 6.3, H-15), 0.89 (3H, s, H-19), 2.03 (3H, s, OAc), 3.35(2H, dd, J₁=92.2, J₂=10.9, 3.56-3.66 (1H, m, H-3). ¹³C NMR (101 MHz,CD₃OD): 6 72.01, 71.74, 41.80, 40.57, 39.96, 38.21, 38.15, 36.46, 35.80,35.17, 34.98, 30.79, 27.46, 26.13, 23.53, 20.49, 15.69, 12.61. IRspectrum (CHCl₃): 3628, 3616 (OH); 2935, 2866 (CH₂); 1380 (CH₃); 1035(C—OH). MS (ESI) in/z: 303.3 (100%, M+Na). HR-MS (ESI) m/z: ForC₁₈H₃₂O₂Na (M+Na) calcd: 303.2295; found: 303.2295.

Example 12(4aS,4bS,7S,8S,8aS,10aR)-7-(Hydroxymethyl)-4a,7,8-trimethyldodecahydrophenanthren-2(1H)-one(14)

Aqueous sodium hypochlorite solution (4.5%, 7.7 ml) was added to asolution of diol 13 (585 mg, 2.09 mmol) in acetic acid (18 ml). Thereaction mixture was stirred at room temperature for 1 h, thenpropan-2-ol (11 ml) was added and the mixture was stirred for 30 min.The reaction was quenched by addition of water (20 ml), the product wasextracted with chloroform (3×50 ml), and the combined organic extractswere washed with saturated aqueous sodium chloride solution and driedover anhydrous sodium sulfate. The solvents were evaporated and theresidue flash chromatographed on silica gel (4-10% acetone in petroleumether). Were obtained 412 mg (71%) of non-crystallising ketone 14:[α]_(D) ²⁰−2.7 (c 0.29, CHCl₃). ¹H NMR (400 MHz, CDCl₃): δ 0.73 (3H, s,H-18), 0.82 (3H, d, J=6.3, H-15), 0.99 (3H, s, H-19), 3.38 (2H, dd,J₁=101.4, J₂=10.9, H-17). ¹³C NMR (101 MHz, CDCl₃): δ 213.44, 71.47,44.04, 42.38, 40.44, 40.22, 38.18, 37.87, 37.40, 36.89, 35.68, 35.34,26.93, 25.51, 22.83, 20.80, 15.72, 12.56. IR spectrum (CHCl₃): 3630(OH); 2935, 2860 (CH₂); 1708 (C═O); 1383 (methyl); 1032 (CCO). MS (ESI)m/z: 301.2 (100%, M+Na). HR-MS (ESI) m/z: For C₁₈H₃₀O₂Na (M+Na) calcd:301.2138; found 301.2139.

Example 13((4aS,4bS,7S,8S,8aS,10aR)-4a,7,8-Trimethyldodecahydro-1H-spiro[phenanthren-2,2′-[1,3]dioxolan]-7-yl)methanol(15)

A mixture of ketone 14 (550 mg, 1.98 mmol), triethyl orthoformate (2.3ml, 13.85 mmol), ethylene glycol (2.2 ml, 39 mmol) and p-toluenesulfonicacid (60 mg, 0.32 mmol) in benzene (10 ml) was stirred at roomtemperature for 1 h. The reaction mixture was then allowed to stand 17 hat 50° C. After cooling, the mixture was poured into saturated aqueoussodium chloride solution, the product was extracted with ethyl acetate,and the combined organic extracts were washed with saturated aqueoussodium chloride solution and dried over anhydrous magnesium sulfate. Thesolvents were evaporated and the residue chromatographed on silica gel(1% triethylamine and 10% ethyl acetate in petroleum ether) yielding 542mg (85%) of oily ketal 15: [α]_(D) ²⁰−2.7 (c 0.26, CHCl₃). ¹H NMR (400MHz, CDCl₃): δ 0.70 (3H, s, H-18), 0.78 (3H, d, J=6.3, H-15), 0.92 (3H,s, H-19), 335 (2H, dd, J₁=89.0, J₂=10.9, H-17), 3.93 (4H, OCH₂CH₂O). ¹³CNMR (101 MHz, CDCl₃): δ 110.20, 71.76, 64.37, 64.22, 40.60, 40.59,39.28, 38.15, 38.00, 35.78, 35.66, 34.99, 34.00, 30.32, 27.02, 25.90,23.27, 20.69, 15.69, 12.58. IR spectrum (CHCl₃): 3630 (OH); 2976, 2881,1381 (methyl); 2928, 1471 (CH₂); 1471, 1094, 947 (ketal); 1030 (COH). MS(CI) m/z: 321.2 (52%, M−H), 323.2 (54%, M+H). HR-MS (CI) m/z: ForC₂₀H₃₃O₃ (M−H) calcd: 321.2433; found: 321.2430.

Example 14(4aS,4bS,7S,8aS,10aR)-7-(Methoxymethyl)-4a,7,8-trimethyldodecahydrophenanthren-2(1H)-one(16)

Sodium hydride (60% suspension in oil, 346 mg) was added to a solutionof the ketal 15 (430 mg, 1.33 mmol) in dried tetrahydrofuran (30 ml) andthe mixture was stirred under an inert atmosphere of argon for 1 h andheated at 90° C. Methyl iodide (0.7 ml, 11.4 mmol) was added and themixture was stirred and heated to 90° C. under an inert atmosphere ofargon for 5 h. After cooling, the product was extracted with ethylacetate; the combined organic extracts were washed with saturatedaqueous sodium chloride solution, dried over anhydrous magnesium sulfateand the solvents evaporated under reduced pressure. The residue wasdissolved in acetone (10 ml), diluted hydrochloric acid (5%, 150 ml) wasadded and the mixture was stirred at room temperature for 1 h. Thereaction was quenched by addition of saturated aqueous sodiumbicarbonate (10 ml), the product was extracted with ethyl acetate, andthe combined organic extracts were washed with brine and dried withmagnesium sulfate. Evaporation of the solvent afforded 322 mg (83%) ofnon-crystallising keto derivative 16: [α]_(D) ²⁰−3.5 (c 0.37,CHCl₃/MeOH, 2: 0.14). ¹H NMR (400 MHz, CDCl₃): δ 0.72 (3H, s, H-19),0.80 (3H, d, J=6.3, H-15), 0.98 (3H, s, H-18), 3.09 (2H, dd, J₁=129.6,J₂=9.1, H-17), 3.32 (3H, OCH₃). ¹³C NMR (101 MHz, CDCl₃): δ 213.49,81.78, 59.45, 44.12, 42.39, 40.75, 40.11, 37.76, 37.40, 36.94, 36.34,35.32, 29.85, 26.94, 25.44, 22.80, 20.85, 15.99, 12.59. IR spectrum(CHCl₃): 2928 (CH₂); 1707 (C═O); 1382 (methyl); 1101 (COC). MS (CI) m/z:293.2 (72%, M+H). HR-MS (CI) In/z: For C₁₉H₃₃O₂ (M+H) calcd: 293.2481;found: 293.2477.

Example 15(2R,4aS,4bS,7S,8aS,10aR)-7-(Methoxymethyl)-4a,7,8-trimethyltetradecahydro-phenanthren-2-ol(17)

A solution of ketone 16 (400 mg, 1.37 mmol) in methanol (20 ml) wascooled to 0° C. and sodium borohydride (57 mg, 1.51 mmol) was addedwhile stirring. The mixture was stirred for 1 h at 0° C., then waswarmed to room temperature and quenched with dilute hydrochloric acid(5%, 15 ml). The product was extracted with chloroform (3×20 ml), thecombined organic extracts were washed with saturated aqueous sodiumbicarbonate solution and dried over anhydrous magnesium sulfate.Evaporation of the solvents and chromatography of the residue (7%acetone in petroleum ether) gave 323 mg (80%) of hydroxy derivative 17:mp 107-108° C. (acetone/n-heptane), [α]_(D) ²⁰−9.2 (c 0.37, CHCl₃). ¹HNMR (400 MHz, CDCl₃): 0.69 (3H, s, H-19), 0.77 (3H, d, J=6.4, H-15),0.88 (3H, s, H-18), 3.07 (2H, dd, J₁=11.3, J₂=9.1, H-17), 3.32 (3H,OCH₃), 3.57-3.66 (1H, m, H-3). ¹³C NMR (101 MHz, CDCl₃): δ 82.11, 72.04,59.45, 41.85, 40.93, 39.88, 38.11, 37.71, 36.48, 36.46, 35.20, 34.96,30.78, 27.48, 26.06, 23.52, 20.54, 15.96, 12.68. IR spectrum (CHCl₃):3609 (OH); 2977 (CH₃); 1100 (COC). MS (ESI) m/z: 317.2 (100%, M+Na).(ESI) m/z: For C₁₉H₃₄O₂Na (M+Na) calcd: 317.2451; found: 317.2451.

Example 16 Pyridinium(2R,4aS,4bS,7S,8S,8aS,10aR)-7-(methoxymethyl)-4a,7,8-trimethyltetra-decahydrophenanthren-2-yl2-sulfate (18)

Compound 18 was prepared according to General Procedure I—Preparation ofC-3 Sulfate from compound 17 (78 mg, 0.26 mmol) affording sulfate 18 (40mg, 33%) : [α]_(D) ²⁰+4.0 (c 0.30, CHCl₃/MeOH, 1.849:0.341). ¹H NMR (400MHz, CDCI₃): δ 0.68 (3H, s, H-19), 0.75 (3H, d, J=6.1, H-15), 0.86 (3H,s, H-18), 3.06 (2H, dd, J₁=112.6, J₂=9.1, H-17), 3.31 (3H, s, OCH₃),4.46 (1H, tt, J₁=10.9, J₂=4.9, H-3), 8.01 (2H, m, H-2′ and H-4′,pyridinium), 8.49 (1H, t, J=8.6, H-3′, pyridinium), 8.92-9.05 (2H, m,H-1′ and H-5′, pyridinium). ¹³C NMR (101 MHz, CDCl₃) 6 145.84 (C-1′,C-5′), 142.41 (C-3′), 127.30 (C-2′, C-4′), 82.13, 79.89, 59.43, 41.84,41.00, 39.82, 38.04, 37.70, 36.50, 35.10, 34.84, 33.25, 27.91, 27.31,26.01, 23.43, 20.50, 15.95, 12.66. IR spectrum (CHCl₃): 2976, 2933(CH₂OCH₃); 1263, 1255, 1183, 1044, 954 (SO₃). MS (ESI) in/z: 373.2(100%, M−H-pyridine). HR-MS (ESI) m/z: For C₁₉H₃₃O₅S (M−H-pyridine)calcd: 373.2054; found: 373.2054.

Example 174-(((2R,4aS,4bS,7S,8aS,10aR)-7-(Methoxymethyl)-42,7,8-trimethyltetradecahydro-phenanthren-2-yl)oxy)-4-oxobutanoicAcid (19)

Compound 19 was prepared according to General Procedure II—preparationof C-3 Hemisuccinate from compound 17 (70 mg, 0.23 mmol). Chromatographyon silica gel (10% acetone in petroleum ether) afforded 78 mg (83%) ofthe derivative 19: [α]_(D) ²⁰+6.5 (c 0.27, CHCl₃).¹H NMR (400 MHz,CDCI₃): δ 0.68 (3H, s, H-19), 0.77 (3H, d, J=6.1, H-15), 0.88 (3H, s,H-18), 2.51-2.76 (4H, m, OCCH₂CH₂CO), 3.08 (2H, dd, J₁=126.8, J₂=9.0,H-17), 3.33 (3H, s, OCH₃), 4.75 (1H, tt, J₁=11.3, J₂=4.8, 11-3). ¹³C NMR(101 MHz, CDCl₃): δ 177.20, 171.85, 81.97, 75.13, 59.44, 41.63, 40.80,39.85, 38.03, 37.71, 36.45, 34.97, 34.85, 32.06, 29.45, 29.05, 27.29,26.67, 25.97, 23.45, 20.55, 16.00, 12.63. IR spectrum (CHCl₃): 2935(CH₂OCH₃); 1717 (C═O, COOH); 1100 (COC). MS (ESI) m/z; 393.3 (100%,M−H). HR.-MS (ESI) m/z: For C₂₃H₃₇O₅ (M−H) calcd: 393.2647; found:393.2643. For C₂₃H₃₈O₅ (394.3) calcd: 70.02% C, 9.71% H; found: 69.76%C, 9.68% H.

Example 18 Methyl (1S,2S,4aS,4bS,7R,8aR,10aS)-7-hydroxy-1,2,4b-trimethyltetradecahydro-phenanthren-2-carboxylate (20)

To a solution of 12 (100 mg, 0.29 mmol) in methanol (5 ml) was addedpotassium hydroxide (60 mg, 1.07 mmol) in methanol (2 ml) and themixture was stirred at ambient for 18 h. Then, it was poured into water;the product was extracted with ethyl acetate. The combined organics werewashed with dilute hydrochloric acid (5%), water, saturated sodiumbicarbonate solution and dried over anhydrous sodium sulfate.Evaporation of solvents under reduced pressure gave a residue of 20 (83mg, 94%) which crystallized from ethyl acetate/n-heptane: mp 144-146° C.(ethyl-acetate/n-heptane), [α]_(D) ²⁰+6.2 (c 0.33, CHCl₃). ¹H NMR (400MHz, CD₃OD): δ 0.73 (3H, d, J=6.7, H-15), 0.89 (3H, s, H-19), 1.06 (3H,s, H-18), 3.67 (3H, s, OCH₃), 3,58-3.68 (1H, H-3). ¹³C NMR (101 MHz,CD₃OD): δ 179.26, 71.92, 51.87, 47.78, 42.32, 41.78, 39.69, 37.81,37.06, 36.43, 35.18, 34.86, 30.73, 27.25, 25.62, 23.41, 20.10, 15.38,14.66. IR spectrum (CHCl₃): 3609, 1054, 1033 (OH); 3020, 2942 (CH₃);1720 (C═O); 1243 (COC). MS (ESI) m/z: 331.3 (100%, M+Na). HR-MS (ESI)m/z: For C,₁₉H₃₂O₃Na (M+Na) calcd: 331.2244, found: 331.2243.

Example 194-(((2R,4aS,7S,8S,10aR)-7-(Methoxykarbonyl)-4a,7,8-trimethyltetradecahydro-phenanthren-2-yl)oxy)-4-oxobutanoicAcid (21)

Compound 21 was prepared according to General Procedure II—Preparationof C-3 Hemisuccinate from compound 20 (100 mg, 0.32 mmol).Chromatography on silica gel (10% acetone in petroleum ether) gavecompound 21 (68 mg, 51%) as a white solid: mp 145-147° C. (ethylacetate/n-heptane), [α]_(D) ²⁰+21.5 (c 0.21, CHCl₃). ¹H NMR (400 MHz,CD₃OD): δ 0.73 (3H, d, J=6.7, H-15), 0.90 (3H, s, H-19), 1.06 (3H, s,H-18), 2.55-2.72 (4H, m, OCCH₂CH₂CO), 3.67 (3H, s, OCH₃), 4.70-4.81 (1H,m, H-3). ¹³C NMR (101 MHz, CD₃OD): δ 17930, 171.91, 74.93, 51.88, 47.77,42.33, 41.58, 39.67, 37.74, 37.03, 34.87, 34.83, 32.11, 29.83, 29.47,29.03, 27.06, 26.70, 25.51, 23.34, 20.09, 15.34, 14.63. IR spectrum(CHCl₃): 3020, 1361 (CH₃); 2950 (CH₂); 1724, 1718 (C═O); 1243, 1166(COC). MS (ESI) m/z: 431.2 (100%, M+Na). HR-MS (ESI) m/z: For C₂₃H₃₆O₆Na(M+Na) calcd: 431.2404; found: 431.2403.

Example 20 Pyridinium(2R,4aS,7S,8S,10aR)-7-(methoxycarbonyI)-4a,7,8-trimethyltetra-decahydrophenanthren-2-yl2-sulfate(22)

Compound 22 was prepared according to General Procedure I—Preparation ofC-3 Sulfate from compound 20 (104 mg, 0.34 mmol) affording sulfate 22(55 mg, 35%): mp 145-147° C. (ethyl-acetate/n-heptane), [α]_(D) ²⁰+10.9(c 0.40, CHCl₃). ¹H NMR (400 MHz, CD₃OD): δ 0.71 (3H, d, J=6.6, H-15),0.88 (3H, s, H-19), 1.05 (3H, s, H-18), 3.67 (3H, s, OCH₃), 4.47 (1H,tt, J₁=10.9, J₂=5, H-3), 8.02 (2H, m, H-2′ and H-4′, pyridinium), 8.49(1H, t, J=7.8, H-3′, pyridinium), 8.96-9.02 (2H, m, H-1″ a H-5′,pyridinium). ¹³C NMR (101 MHz, CD₃OD): δ 179.30, 145.89 (C-1′, C-5′),142.41, 127.33 (C-2′, C-4′), 79.53, 51.85, 47.75, 42.37, 41.77, 39.62,37.72, 37.09, 35.10, 34.73, 33.27, 27.91, 27.07, 25.55, 23.32, 20.06,15.36, 14.64. IR spectrum (CHCl₃): 3140, 3100, 1490 (pyridinium); 1720(C═O); 1264, 1183, 1175, 1044, 954 (SO₃); 1245, 1192 (C—O). MS (ESI)m/z: 387.2 (100%, M-pyridine). HR-MS (ESI) m/z: For C₁₉H₃₁O₆S(M-pyridine) calcd: 387.1847, found: 387.1844.

Example 21 2S,4aS,4bS,7S,8aR,10aR)-7-hydroxy-2,4b-dimethyl-1-(((E)-3-oxoindolin-2-yliden)-methyl)tetradecahydrophenanthren-2-carboxylicAcid (24)

A solution of o-nitrobenzaldehyde (2.75 g) in methanol (25 ml) was addedto a mixture of 3beta-hydroyx-5beta-androstan-17-one (23) (5.0 g, 17.22mmol) in methanolic potassium hydroxide solution (4%, 125 ml). Thereaction mixture was stirred at room temperature for 18 h and methanolsolution of potassium hydroxide (0.5 g in 1 ml) and o-nitrobenzaldehyde(275 mg in 2.5 ml) were added. After 20 h, the reaction mixture wasconcentrated under reduced pressure to ⅓ of volume, diluted with water(20 m), filtered over active carbon and the filtrate was acidified withdilute hydrochloric acid (5%). The yellow precipitated solids of 24 wereisolated, washed with water and dried (6.1 g, 84%): mp 242-245° C.(metanol/water), [α]_(D) ²⁰−182.2 (c 0.29, CH₃OH). ¹H NMR (400 MHz,CD₃OD): δ 0.98 (3H, s, H-18), 1.24 (3H, s, H-19), 2.86 (1H, dd, J₁=11.4,J₂=10, H-14), 4.02-4.07 (1H, m, H-3), 5.81 (1H, d, J=11.6, H-15), 6.79(1H, t, J=7.7), 6.93 (1H, d, J=8.2), 7.41(1H, t, J=7.7), 7.52 (1H, d,J=7.7). ¹³C NMR (101 MHz, CD₃OD): δ 187.72, 18139, 155.99, 140.14,137.88, 125.48, 121.86, 119.96, 117.67, 112.83, 67.68, 48.85, 48.09,39.98, 38.12, 37.64, 37.60, 36.53, 34.23, 30.63, 28.55, 27.74, 27.64,24.26, 21.01, 16.09. IR spectrum (KBr): 3445, 3343 (OH, NH); 2976, 1383(methyl); 2613 (OH, dimer); 1708 (C═O, dimer); 1693 (C═O, indolone);1639 (C═C); 1613, 1486, 1468, 1448 (ring, indolone); 1003 (C—OH). MS(ESI) m/z: 446.3 (100%, M+Na), 424.3 (45%, M+H). HR-MS (ESI) m/z: ForC₂₆H₃₃O₄NNa (M+Na) calcd; 446.2302; found: 446.2301.

Example 22 Methyl(2S,4aS,4bS,7S,8aR,10aR)-7-hydroxy-2,4b-dimethyl-1-(((E)-3-oxoindolin-2-yliden)methyl)-tetradecahydrophenanthren-2-carboxylate(25)

A freshly prepared ethereal solution of diazomethane was added whilestirring to a cooled solution of compound 24 (0° C., 5 g, 11.80 mmol) inether. After completion of reaction (TLC), excess of diazomethane wasevaporated affording 5.1 g (99%) of non-crystallising methyl ester (25):[α]_(D) ²⁰−208.1 (c 0.26, CHCl₃). ¹H NMR (400 MHz, CDCl₃): δ 0.97 (3H,s, H-18), 1.24 (3H, s, H-19), 2.72 (1H, t, J=10.7, H-14), 3.56 (1H, s,OCH₃), 4.12-4.14 (1H, m, H-3), 5.77 (1H, d, J=11.1, H-15), 6.87 (1H, t,J=7.7), 6.93 (1H, d, J, 8.2), 7.42 (1H, t, J=7.7), 7.66 (1H, d,J=7.7).¹³C NMR (101 MHz, CDCl₃): δ 185.93, 179.78, 154.04, 138.80,136.25, 125.06, 122.45, 119.89, 116.52, 112.22, 67.03, 52.44, 47.65,47.54, 38.67, 37.81, 36.59, 36.33, 35.60, 33.58, 29.64, 28.00, 26.94,26.51, 23.85, 19.91, 16.00. IR spectrum (CHCl₃): 3616 (OH); 2979, 1435,1384 1716 (C═O); 1701 (C═O, indolone); 1644 (C═C). MS (ESI) m/z: 460.4(100%, M+Na), 438.4 (52%, M+H). HR-MS (ESI) m/z: For C₂₇H₃₆O₄N (M+H)calcd: 438.2639; found: 438.2639.

Example 23 Methyl(2S,4aS,4bS,7S,8aR,10aR)-7-acetoxy-2,4b-dimethyl-1-(((E)-3-oxoindolin-2-yliden)methyl)-tetradecahydrophenanthren-2-carboxylate(26)

To a cold (0° C.) mixture of methyl ester 25 (7.2 g, 16.45 mmol) and4-(N,N-dimethylamino)pyridine (200 mg, 1.64 mmol) in pyridine (50 ml)was added acetic anhydride (46 ml). The reaction mixture was warmed toroom temperature and after 2 h; the reaction was quenched by adding asmall amount of water. The reaction mixture was poured into dilutehydrochloric acid (5%, 150 ml), the product was extracted with ethylacetate (3×80 ml), and the combined organic phases were washed withwater (2×100 ml), saturated sodium bicarbonate, and saturated brine anddried with anhydrous magnesium sulfate. The solvents were evaporatedunder reduced pressure yielding 7.0 g (89%) of non-crystallizingcompound 26: [α]_(D) ²⁰−221.1 (c 0.274, CHCl₃). ¹H NMR (400 MHz, CDCl₃):δ 0.98 (3H, s, H-18), 1.24 (3H, s, H-19), 2.04 (3H, s, OAc), 2.73 (1H,t, J=10.7, H-14), 3.56 (3H, s, OCH₃), 5.05-5.11 (1H, m, H-3), 5.76 (1H,d, J=11.2, H-15), 6.87 (1H, t, J=7.7), 6.93 (1H, d, J=8.2), 7.42(1H, t,J=7.7), 7.65 (1H, d, J=7 .7). ¹³C NMR (101 MHz, CDCl₃): δ 185.92,179.66, 170.81, 154.04, 138.83, 136.28, 125.04, 122.43, 119.90, 116.31,112.23, 70.55, 52.44, 47.61, 38.82, 47.46, 37.74, 37.14, 36.60, 35.35,30.63, 30.46, 26.82, 26.36, 25.18, 23.80, 21.62, 19.90, 16.00. IRspectrum (CHCl₃): 2979, 1385, 1379 (methyl); 1727 (C═O, OAc); 1717 (C═O,COOMe); 1702 (C═O, indolone); 1645 (C═C); 1615, 1485, 1470, 1448 (ring,indolone). MS (ESI) m/z: 502.4 (100%, M+Na), 480.4 (30%, M+H). HR-MS(ESI) m/z: For C₂₉H₃₈O₅ (M+H) calcd: 480.2745; found: 480.2746.

Example 24 Methyl(2S,4aS,4bS,7S,8aR,10aR)-7-acetoxy-1-formyl-2,4b-dimethyltetradecahydrophenanthren-2-carbaxylate(27)

Compound 27 was prepared from compound 26 (7.0 g, 14.60 mmol)analogously to the preparation of the substance 11 using dimethylsulfide (3.4 ml, 46.53 mmol, 1 h, room temperature). Chromatography onsilica gel (180 g, 6% ethyl acetate in petroleum ether) afforded 4.27 g(80%) of non-crystallising aldehyde 27, which was used crude in the nextreaction :¹H NMR (400 MHz, CDCl₃): 0.99 (3H, s, H-18), 1.24 (3H, s,H-19), 2.04 (3H, s, OAc), 2.62 (1H, dd, J₁=11.2 and J₂=3.0, H-14), 3.69(3H, s, OCH₃), 5.05-5.11 (1H, m, H-3), 9.72 (1H, d, J=3.0, CHO). ¹³C NMR(101 MHz, CDCl₃): δ 204.77, 177.45, 170.75, 70.45, 60.18, 52.32, 38.45,45.24, 36.97, 36.85, 35.27, 32.41, 30.58, 30.51, 2633, 26.26, 25.10,23.71, 21.61, 19.73, 16.89.

Example 25 Methyl (2S,4aS,4bS,7S,8aR,10aS)-7-acetoxy-2,4b-dimethyltetradecahydrophenanthren-2-carboxylate (28)

Compound 28 was prepared according to General Procedure VI—WilkinsonDecarbonylation of compound 27 (2 g, 5.49 mmol). Chromatography onsilica gel (3% acetone in petroleum ether) afforded 1.31 g (71%, oil) ofcompound 28:[α]_(D) ²⁰+14.6 (c 0.314, CHCl₃). ¹H NMR (400 MHz, CDCl₃): δ0.95 (3H, s, H-18), 1.19 (3H, s, H-19), 2.05 (3H, s, H-OAc), 3.66 (3H,s, H-OCH₃), 5.03-5.10 (1H, m, H-3). ¹³C NMR (101 MHz, CDCl₃): δ 179.37,170.84, 70.79, 51.89, 42.43, 39.41, 37.56, 34.97, 34.49, 31.73, 30.65,30.32, 28.82, 26.53, 25.21, 23.93, 21.65, 20.57, 20.51. IR spectrum(CHCl₃): 2975, 2941, 1491, 1378 (methyl); 1722 (C═O); 1262, 1243, 1025(C—O). MS (ESI) m/z: 359.2 (100%, M+Na). HR-MS (ESI) m/z: For C₂₀H₃₂O₄Na(M+Na) calcd: 359.2193; found: 359.2191.

Example 26(2S,4aS,4bS,7S,8aS,10aR)-7-(Hydroxymethyl)-4a,7-dimenthyltetradecahydrophenanthren-2-ol(29)

Compound 29 was prepared from compound 28 (1.25 g, 3.71 mmol)analogously to the preparation of the compound 13 to give 905 mg (92%)of the diol 29: mp 139-140° C. (ethyl-acetate/n-heptane), [α]_(D)²⁰+13.1 (c 0.29, CHCl₃). ¹H NMR (400 MHz, CDCl₃): δ 0.89 (3H, s, H-18),0.95 (3H, s, H-19), 3.24-3.30 (2H, m, CH₂OH), 4.08-4.14 (1H, m, H-3).¹³C NMR (101 MHz, CDCl₃): δ 74.89, 67.29, 42.71, 39.88, 39.08, 35.85,35.34, 34.37, 33.59, 31.92, 29.62, 29.34, 28.10, 26.79, 24.09, 20.74,20.32. IR spectrum (CHCl₃): 3630, 3618 (OH); 2936, 2863 (CH₂); 1031, 998(C—OH). MS (ESI) m/z: 289.2 (100%, M+Na). HR-MS (CI) m/z: For C₁₇H₂₉O₂(M−H) calcd: 265.2168; found: 265.2170.

Example 27(2S,4aS,4bS,8aR,10aS)-2,4b-Dimethyl-7-oxotetradecahydrophenanthren-2-carbaldehyde(30)

Anhydrous sodium acetate (158 mg, 1.93 mmol) and pyridiniumchlorochromate (826 mg, 3.86 mmol) were added to a solution of compound29 (320 mg, 1.20 mmol) in dichloromethane (20 ml). The reaction mixturewas stirred at room temperature under an inert atmosphere of argon for 2h and then it was diluted with ethyl acetate (80 ml) and filteredthrough a column of neutral alumina (60 g). The solvents were evaporatedunder reduced pressure to obtain 280 mg (89%) of aldehyde 30, which wasused crude in the next reaction: ¹H NMR (400 MHz, CDCl₃): δ 1.02 (3H, s,H-19), 1.12 (3H, s, H-18), 9.41 (1H, s, CHO). ¹³C NMR (101 MHz, CDCI₃):δ 212.89, 206.05, 44.45, 42.37, 40.38, 39.05, 37.35, 36.57, 35.11,31.29, 31.17, 29.85, 28.45, 26.60, 22.81, 20.02, 17.58.

Example 28(4aS,4bS,7R,8aS,10aR)-4a,7-Dimethyldodecahydrophenanthren-2(1H)-one(31a),(4aS,4bS,8aS,10aR)-4a,7-dimethyl-3,4,4a,4b,5,8,8a,9,10,10a-decahydrophenanthren-2(1H)-one(31b) and(4aS,4bS,8aS,10aR)-4a,7-dimethyl-3,4,4a,4b,5,6,8a,9,10,10a-decahydrophenanthren-2(1H)-one(31c)

Compounds 31a, 31b, and 31c were prepared according to General ProcedureVI—Wilkinson Decarbonylation of compound 30 (280 mg, 1.07 mmol).Chromatography on silica gel (3% acetone in petroleum ether) gave aninseparable mixture of three products 31a, 31b, and 31c (210 mg,91.1:4.3:4.6).

Example 29(2R,4aS,4bS,7R,8aS,10aR)-4a,7-Dimethyldodecahydrophenanthren-2-ol (32a),(2R,4aS,4bS,8aS,10aR)-4a,7-dimethyl-3,4,4a,4b,5,8,8a,9,10,10a-decahydrophenanthren-2-ol(32b) and(2R,4aS,4bS,8aS,10aR)-4a,7-dimethyl-3,4,4a,4b,5,6,8a,9,10,10a-decahydrophenanthren-2-ol(32c)

A mixture of three compounds 31a, 31b and Mc in tetrahydrofuran (18 ml)was cooled to −40° C. and tri-tert-butoxy lithium aluminum hydride (210mg, 0.83 mmol) was added while stirring. After 2 h, the mixture waswarmed to room temperature and quenched with aqueous hydrochloric acid(5%, 20 ml). The product was extracted with chloroform, the combinedorganic extracts were washed with saturated sodium bicarbonate,saturated sodium chloride solution and dried over anhydrous sodiumsulfate. The solvents were evaporated under reduced pressure to give amixture of the three hydroxy derivatives 32a, 32b and 32c (180 mg).

Example 30(2R,4aS,4bS,7R,8aS,10aR)-4a,7-Dimethyltetradecahydrophenanthren-2-ol(33)

To the residue of compounds 32a, 321, and 32c dissolved indichloromethane (15 ml) was added a mixture of sodium acetate (66 mg,0.81 mmol), water (0.6 ml), and acetic peroxacetic acid (9%, 2.4 ml) andthe reaction mixture was stirred for 2 h room temperature. The reactionwas quenched by addition of saturated aqueous sodium sulfite solutionand the product was extracted with chloroform, the combined organicextracts were washed with water and dried over anhydrous magnesiumsulfate. The solvents were evaporated under reduced pressure andchromatography (5% acetone in petroleum ether) afforded 90 mg (36%, 3steps) of desired compound 33: [α]_(D) ²⁰+24.0 (c 0.27, CHCl₃). ¹H NMR(400 MHz, CDCl₃): δ 0.79 (3H, s, H-19), 0.84 (3H, d, J=7.3, H-18), 3.53(1H, tt, J₁=11.1 and J₂=4.7, H-3). ¹³C NMR (101 MHz, CDCl₃): δ 72.11,42.49, 41.02, 40.80, 36.58, 34.86, 34.81, 32.70, 30.95, 30.87, 29.48,27.81, 27.37, 23.62, 19.35, 18.31. IR spectrum (CHCl₃): 3608 (OH); 2958,2866 (methyl); 1038, 1029 (C—OH). MS (CI) m/z: 236.2 (8%, M), 235.2(15%, M−H). HR-MS (CI) m/z: For C₁₆H₂₇O (M−H) calcd: 235.2062; found:235.2070.

Example 31 4(((2R,4aS,4bS,7R,8aS,10aR)-4a,7-Dimethyltetradecahydrophenanthren-2-yl)oxy)-4-oxobutanoic acid (34)

Compound 34 was prepared according to General Procedure II—Preparationof C-3 Hemisuccinate from compound 33 (65 mg, 0.27 mmol). Chromatographyon silica gel (10% acetone in petroleum ether) afforded 40 mg (43%) of34: mp 119-121° C. (acetone/n-heptane), [α]_(D) ²⁰+43.9 (c 0.273,CHCl₃). ¹H NMR (400 MHz, CDCI₃): δ 0.90 (3H, s, H-18), 0.94 (3H, d, J,7.2, H-19), 2.74-2.54 (4H, m, H-2′ a H-4′), 4.75 (1H, tt, J₁=11.4 andJ₂=4.7, H-3). ¹³C NMR (100 MHz, CDCl₃): δ 171.26, 171.83, 75.29, 42.32,40.99, 40.78, 34.84, 34.53, 32.67, 32.33, 30.92, 29.45, 29.40, 29.06,27.80, 27.20, 26.89, 23.58, 19.36, 18.31. IR spectrum (CHCl₃): 1727,1716 (C═O); 1280 (C—O, dimer); 1232, 1176 (COC). MS (ESI) m/z: 335.4(100%, M−H). HR-MS (ESI) m/z: For C₂₀H₃₁O₄ (M−H) calcd: 335.2228; found:335.2228.

Example 32 Pyridinium(2R,4aS,4bS,7R,8aS,10aR)-4a,7-dimethyltetradecahydrophenanthren-2-yl2-sulfate (35)

Compound 35 was prepared according to General Procedure I—Preparation ofC-3 Sulfate from compound 33 (108 mg, 0.46 mmol) affordning sulfate 35(66 mg, 36%): mp 147-149° C., [α]_(D) ²⁰+25.3 (c 0.25, CHCl₃). ¹H NMR(400 MHz, CDCl₃): δ 0.88 (3H, s, H-18), 0.92 (3H, d, J=7.2, H-19), 4.46(1H, tt, J₁=11.3 and J₂=4.8, H-3), 8.00 (2H, m, H-2′ and H-4′,pyridinium), 8.47 (1H, t, J=8.6, H-3′, pyridinium), 8.95-9.01 (2H, m,H-1′ and H-5′, pyridinium). ¹³C NMR (100 MHz, CDCl₃): δ 145.78 (C-1′,C-5′), 142.51 (C-3′), 127.26 (C-2′, C-4′), 79.87, 42.52, 41.06, 40.74,34.81, 34.70, 33.45, 32.72, 30.89, 29.40, 28.05, 27.81, 27.20, 23.54,19.33, 18.30. IR spectrum (CHCl₃): 3140, 3099, 1490, 826 (pyH); 1380(methyl); 1263, 1255, 1173, 1047, 953 (SO₃). MS ESI m/z: 315.3 (100%,M−H-pyridine). HR-MS (ESI) m/z: For C₁₆H₂₇O₄S (M−H-pyridine) calcd:315.1636; found: 315.1634.

Example 33 Methyl(2S,4aS,4bS,7S,8aR,10aS)-7-hydroxy-2,4b-dimethyltetradecahydrophenanthren-2-carboxylate(36)

A mixture of potassium hydroxide (1.3 g, 23.2 mmol) in water (1 ml) andmethanol (40 ml) was added to a solution of acetate 28 (1.7 g, 5.1 mmol)in methanol (85 ml) while stirring. After 7 h of standing at roomtemperature, the reaction mixture was concentrated under reducedpressure to ⅓ of its volume and the product was extracted with ethylacetate (3×20 ml), the combined organic phases were washed with water(2×20 ml), dilute hydrochloric acid (5%, 2×20 ml), saturated sodiumbicarbonate, saturated sodium chloride solution and dried over anhydroussodium sulfate. The solvents were evaporated under reduced pressureyielding 1.24 g (83%) of non-crystallizing compound 36: [α]_(D) ²⁰+13.5(c 0.47, CHCl₃). ¹H NMR (400 MHz, CDCl₃): δ 0.94 (3H, s, H-19), 1.19(3H, s, H-18), 3.66 (3H, s, H—OCH₃), 4.07-4.15 (1H, m, H-3). ¹³C NMR(101 MHz, CDCI₃): δ 179.45, 67.19, 51.87, 42.49, 39.26, 36.76, 35.22,34.53, 33.54, 31.71, 29.50, 28.96, 28.06, 26.68, 23.97, 20.57, 20.51. IRspectrum (CHCl₃): 1719 (C═O); 1381 (methyl); 1245, 1033 (C—O). MS ESI)m/z: 317.2 (100%, M+Na). HR-MS (ESI) m/z: For C₁₈H₃₀O₃Na (M+Na) calcd:317.2087; found: 317.2087.

Example 34 Methyl(2S,4aS,4bS,8aR,10aS)-2,4b-dimethyl-7-oxotetradecahydrophenanthren-2-earboxylate(37)

Jones reagent was added dropwise to a cooled (0° C.) solution of thehydroxy derivative 36 (300 mg, 1.02 mmol) in acetone (5 ml). Theprogress of reaction was followed by TLC and quenched with methanol (5ml). The mixture was poured into water, the product was extracted withethyl acetate (3×20 ml), the combined organic phases were washed withsaturated sodium bicarbonate solution, water and dried over anhydroussodium sulfate. The solvents were evaporated under reduced pressureyielding 250 mg (84%) of ketone 37: [α]_(D) ²⁰+22.9 (c 0.38, CHCl₃). ¹HNMR (400 MHz, CDCl₃): δ 1.00 (3H, s, H-19), 1.22 (3H, s, H-18), 3.66(3H, s, OCH₃). ¹³C NMR (101 MHz, CDCI₃): δ 213.04, 179.09, 51.93, 44.51,42.38, 42.24, 40.24, 37.38, 36.61, 35.02, 34.38, 31.58, 29.83, 28.38,26.66, 22.75, 20.60, 20.55. IR spectrum (CHCl₃): 2980, 2934, 1465, 1435,1383 (methyl); 1720, 1711 (C═O); 1244, 1124 (C—O). MS (ESI) m/z: 315.2(100%, M+Na). HR-MS (ESI) m/z: For C₁₈H₂₈O₃Na (M+Na) calcd: 315.1931;found: 315.1929.

Example 35 Methyl(2S,4aS,4bS,7R,8aR,10aS)-7-hydroxy-2,4b-dimethyltetradecahydrophenanthren-2-carboxylate(38)

Compound 38 was prepared from compound 37 (100 mg, 0.34 mmol)analogously to the preparation of compound 32 affording crystallisinghydroxy derivative 38 (71 mg, 71%): [α]_(D) ²⁰+21.6 (c 0.19, CHCl₃). ¹HNMR (400 MHz, CDCl₃): δ 0.90 (3H, s, H-18), 1.18 (3H, s, H-19), 3.66(3H, s, H—OCH₃), 3.58-3.65 (1H, m, H-3). ¹³C NMR (101 MHz, CDCl₃): δ179.37, 171.94, 51.88, 42.39, 42.33, 39.98, 36.52, 34.91, 34.66, 34.48,31.91, 30.82, 29.10, 27.22, 23.46, 20.59, 20.33. IR spectrum (CHCl₃):3608 (OH); 2976, 2936, 1435, 1389 (methyl); 2936, 2865 (CH₂); 1720(C═O); 1192, 1126, 1036, 1022 (C—O). MS (ESI) m/z: 317.2 (100%, M+Na).HR-MS (ESI) m/z: For C₁₈H₃₀O₃Na (M+Na) calcd: 317.2087; found: 317.2088.

Example 364-(((2R,4aS,4bS,7S,8aS,10aR)-7-(Methoxycarbonyl)-4a,7,8a-trimethyltetradecahydro-phenanthren-2-yl)oxy)-4-oxobutanoicaced (39)

Compound 39 was prepared according to General Procedure II—Preparationof C-3 Hemisuccinate from compound 38 (60 mg, 0.20 mmol). Chromatographyon silica gel (15% acetone in petroleum ether) afforded 42 mg (52%) ofthe derivative 39: mp 110-111° C. (acetone/n-heptane), [α]_(D) ²⁰+23.3(c 0.29, CHCl₃). ¹H NMR (400 MHz, CDCl₃): δ 0.91 (3H, s, H-19), 1.19(3H, s, H-18), 2.56-2.72 (4H, m, H-2′ and H-4′), 3.66 (3H, s, H—OCH₃),4.75 (1H, tt, J₁=11.3, J₂=4.7, H-3). ¹³C NMR (101 MHz, CDCl₃): δ 179.37,176.38, 171.86, 75.02, 51.91, 42.42, 42.39, 42.15, 39.97, 34.70, 34.58,34.48, 32.24, 31.89, 29.46, 29.02, 28.92, 27.06, 26.83, 23.42, 20.58,20.36. IR spectrum (CHCl₃): 2937 (OCH₃); 1718 (C═O); 1242, 1126, 1102(C—O). MS (ESI) m/z: 393.3 (100%, M−H), 394.3 (25%, M). HR-MS (ESI) m/z:For C₂₂H₃₃O₆ (M−1) calcd: 393.2283; found: 393.2283.

Example 37 Methyl(2S,4aS,4bS,7R,8aR,10aS)-2,4b-dimethyl-7-(sulfooxy)tetradecahydrophenanthren-2-carboxylate(40)

Compound 40 was prepared according to General Procedure I—Preparation ofC-3 Sulfate from compound 38 (100 mg, 0.34 mmol) affording sulfate 40(72 mg, 47%): mp 115-118° C., [α]_(D) ²⁰+24.1 (c 0.23, CHCl₃). ¹H NMR(400 MHz, CDCl₃): δ 0.89 (3H, s, H-19), 1.17 (3H, s, H-18), 3.65 (3H, s,OCH₃), 4.45 (1H, tt, J₁=11.0 and J₂=5M, H-3), 8.01 (2H, m, H-2′ andH-4′, pyridinium), 8.48 (1H, tt, J₁=7.9, J₂=1.5, H-3′, pyridinium), 8.99(2H, dd, J₁=6.5, J₂=1.4), H-1′ and H-5′, pyridinium). ¹³C NMR (101 MHz,CDCl₃): δ 179.39, 145.90 (C-1′, C-5′), 142.46 (C-3′), 127.33 (C-2′,C-4′), 79.55, 64.51, 51.87, 42.40, 42.33, 39.91, 34.83, 34.55, 34.48,33.38, 31.84, 29.02, 28.01, 27.05, 23.38, 20.57, 20.29. IR spectrum(CHCl₃): 3140, 3099, 1490 (pyH); 1720 (C═O); 1435, 1380 (methyl); 1254,1049, 957 (SO₃); 1245, 1192, 1013 (C-13 O). MS (ESI) m/z: 373.2 (100%,M−H-pyridine). HR-MS (ESI) m/z: For C₁₈H₂₉O₆S (M−H-pyridine) calcd:373.1690; found: 373.1688.

Example 38(4aS,4bS,7S,8aS,10aR)-7-(Hydroxymethyl)-4a,7-dimethyldodecahydrophenanthren-2(1H)-one(41)

Aqueous sodium hypochlorite solution (4.5%, 1.05 ml) was added to asolution of dial 29 (80 mg, 0.30 mmol) in acetic acid (2.5 ml). Thereaction mixture was stirred at room temperature for 1 h, and then wasadded propan-2-ol (1.5 ml) and the mixture was stirred for 30 min. Thereaction was quenched by addition of water (5 mL), the product wasextracted with chloroform (3×10 ml), and the combined organic extractswere washed with brine and dried with anhydrous magnesium sulfate. Thesolvents were evaporated and the residue flash chromatographed on silicagel (4-10% acetone in petroleum ether) yielding 48 mg (60%) of oilyketone 41: [α]_(D) ²⁰+26.1 (c 0.38, CHCl₃). ¹H NMR (400 MHz, CDCl₃): δ0.92 (3H, s, H-18), 1.01 (3H, s, H-19), 3.24-3.34 (2H, m, CH₂OH). ¹³CNMR (101 MHz, CDCl₃): δ 213.36, 74.67, 44.65, 42.48, 42.47, 40.85,37.42, 36.74, 35.86, 35.14, 34.24, 31.79, 28.77, 26.76, 22.85, 20.84,20.28. IR spectrum (CHCl₃): 3630 (OH); 2931, 2867 (CH₂); 1705 (C═O);1034 (CCO). MS (ESI) m/z: 287.2 (100%, M+Na). HR-MS (ESI) m/z: ForC₁₇H₂₈O₂Na (M+Na) calcd: 287.1982; found: 287.1981.

Example 39((4aS,4bS,7S,8aS,10aR)-4a,7-Dimethyldodecahydro-1H-spiro[phenanthren-2,2′-[1,3]dioxolan]-7-yOmethanol(42)

Compound 42 was prepared from compound 41 (350 mg, 132 mmol) analogouslyto the preparation of compound 15. Chromatography on silica gel (1%triethylamine and 10% ethyl acetate in petroleum ether) gave 336 mg(82%) of oily ketal 42: [α]_(D) ²⁰+19.5 (c 0.11, CHCl₃). ¹H NMR (400MHz, CDCl₃): δ 0.89 (3H, s, H-18), 0.93 (3H, s, H-19), 3.26 (2H, s,CH₂OH), 3.93 (4H, s, OCH₂CH₂O). ¹³C NMR (101 MHz, CDCl₃): δ 110.20,74.94, 64.39, 64.22, 42.70, 41.22, 39.96, 35.82, 35.75, 34.77, 34.39,33.86, 31.90, 30.37, 29.23, 26.86, 23.30, 20.74, 20.27. IR spectrum(CHCl₃): 3630, 3475 (OH); 1382, 1364 (methyl); 1183, 1093, 1068, 947(COCOC); 1034 (CCO). MS (ESI) m/z: 331.3 (100%, M+Na). HR-MS (ESI) m/z:For C₁₉H₃₂O₃Na (M+Na) calcd: 331.2244; found: 331.2246.

Example 40(4aS,4bS,7S,8aS,10aR)-7-(Methoxymethyl)-4a,7-diirnethyldodecahydro-1H-spiro-[phenanthren-2,2′-[1,3]dioxolane](43a)

Sodium hydride (60% suspension in oil, 200 mg) was added to a solutionof the ketal 42 (250 mg, 0.81 mmol) in dried tetrahydrofuran (17 ml) andthe raction mixture was stirred under inert atmosphere of argon for 1 hat 90° C. Methyl iodide was added (0.4 ml, 6.5 mmol) and the mixture washeated under inert atmosphere of argon for 5 h at 90° C. After cooling,the product was extracted with ethyl acetate; the combined organicextracts were washed with saturated aqueous sodium chloride solution anddried over anhydrous magnesium sulfate. Evaporation of the solventafforded 240 mg (92%) of non-crystallising methoxyderivative 43a:[α]_(D) ²⁰+15.9 (c 0.41, CHCl₃). ¹H NMR (400 MHz, CDCl₃): δ 0.89 (3H, s,H-18), 0.92 (3H, s, H-19), 2.99 (2H, s, CH₂), 3.33 (3H, s, OCH₃), 3.93(4H, m, OCH₂CH₂O). ¹³C NMR (100 MHz, CDCl₃): δ 110.24, 85.24, 64.37,64.21, 59.54, 43.20, 41.25, 39.85, 35.75, 35.23, 34.91, 34.76, 33.87,31.87, 30.36, 29.19, 26.89, 23.30, 20.81, 20.73. IR spectrum (CHCl₃):2928, 1449 (CH₂); 2832, 1438, 1387, 1381, 1366 (methyl); 1186, 1094,1068, 947 (COCOC); 1102 (COC), MS (ESI) m/z: 345.3 (55%, M+Na). HR-MS(ESI) m/z: For C₂₀H₃₅O₃(M+H) calcd: 323.2581; found: 323.2580.

Example 41(4aS,4bS,7S,8aS,10aR)-7-(Methoxymethyl)-4a,7-dimethyldodecahydrophenanthren-2(1H)-one(43)

To a solution of ketal 43a (145 mg, 0.45 mmol) in acetone (2.5 ml) wasadded diluted hydrochloric acid (5%, 50 ml) and the mixture was stirredat room temperature for 1 h. The reaction was quenched by addition ofsaturated aqueous sodium bicarbonate solution (10 ml), the product wasextracted with ethyl acetate, and the combined organic extracts werewashed with saturated aqueous sodium chloride solution and dried overanhydrous sodium sulfate. Evaporation of the solvent afforded 120 mg(96%) of non-crystalline keto derivative 43: [α]_(D) ²⁰+24.8 (c 0.42,CHCl₃). ¹H NMR (400 MHz, CDCl₃): δ 0.91 (3H, s, H-18), 1.00 (3H, s,H-19), 3.02 (2H, s, CH₂OH), 3.34 (3H, s, OCH₃). ¹³C NMR (100 MHz,CDCl₃): δ 213.45, 84.91, 59.55, 46.05, 42.93, 42.49, 40.71, 37.43,36.77, 35.27, 35.13, 34.70, 31.77, 28.72, 26.79, 22.85, 20.85, 20.83. IRspectrum (CHCl₃): 2931, 2866 (CH₂); 1706 (C═O); 1383 (methyl); 1102(COC). MS (ESI) m/z: 301.3 (100%, M+Na), 279.3 (20%, M+H). HR-MS (ESI)m/z: For C₁₈H₃₀O₂Na (M+Na) calcd: 301.2138; found: 301.2138.

Example 42(2R,4aS,4bS,7S,8aS,10aR)-7-(Methoxymethyl)-4a,7-dimethyltetradecahydrophenanthren-2-ol(44)

Compound 44 was prepared from compound 43 (40 mg, 0.14 mmol) analogouslyto the preparation of compound 17 by using methanol. Chromatography (10%acetone in petroleum ether) gave 28 mg (70%) of an oily hydroxyderivative 44: [α]_(D) ²⁰+19.7 (c 0.44, CHCl₃). ¹H NMR (400 MHz, CDCl₃):δ 0.88 (3H, s, H-18), 0.89 (3H, s, H-19), 3.00 (2H, s, CH₂OH), 3.33 (3H,s, OCH₃), 3.58-3.67 (1H, m, H-3). ¹³C NMR (101 MHz, CDCl₃): δ 85.22,72.05, 59.55, 43.15, 42.44, 40.48, 36.53, 35.21, 35.00, 34.88, 34.73,32.06, 30.82, 29.40, 27.32, 23.54, 20.79, 20.52. IR spectrum (CHCl₃):3609, 3451 (OH); 2956, 2865, 1388, 1380 (methyl); 1102 (COC); 1036(C—OH). MS (ESI) m/z: 303.4 (100%, M+Na). HR-MS (ESI) in/z: For C₁₈H₃₂O₂Na (M+Na) calcd: 303.2295; found: 303.2295.

Example 434(((2R,4aS,4bS,7S,8aS,10aR)-7-(Methoxymethyl)-4a,7-dimethyltetradecahydrophenanthren-2-yl)oxy)-4-oxobutanoicacid (45)

Compound 45 was prepared according to General Procedure II—Preparationof C-3 Hemisuccinate from compound 44 (60 mg, 0.21 mmol). Chromatographyon silica gel (10% acetone in petroleum ether) afforded 41 mg (50%) ofthe derivative 45: mp 107-109° C. (acetone/n-heptane), [α]_(D) ²⁰+38.8(c 0.26, CHCl₃). ¹H NMR (400 MHz, CDCl₃): δ 0.88 (3H, s, H-19), 0.90(3H, s, H-18), 2.53-2.72 (4H, m, OCCH₂CH₂CO), 3.01 (2H, s, CH₂OH), 3.34(3H, s, OCH₃), 4.69-4.81 (1H, m, H-3). ¹³C NMR (101 MHz, CDCl₃): δ177.12, 171.83, 85.10, 75.17, 59.55, 43.06, 42.26, 40.44, 35.24, 34.78(2×), 34.68, 32.24, 32.04, 29.45, 29.33, 29.04, 27.17, 26.81, 23.50,20.85, 20.55. IR spectrum (CHCl₃): 1726, 1717 (C═O); 1385 (methyl);1169, 1101 (COC). MS (ESI) m/z: 403.2 (100%, M+Na). HR-MS (ESI) m/z: ForC₂₂H₃₆O₅Na (M+Na) calcd: 403.2455; found: 403.2455.

Example 44 Pyridinium(2R,4aS,4bS,7S,8aS,10aR)-7-(methoxymethyl)-4a,7-dimethyltetradecahydrophenanthren-2-yl2-sulfate (46)

Compound 46 was prepared according to General Procedure I—Preparation ofC-3 Sulfate from compound 44 (98 mg, 0.35 mmol). Sulfate 46 was obtained(105 mg, 68%): mp 132-134° C., [α]_(D) ²⁰+22.0 (c 0.26, CHCl₃). ¹H NMR(400 MHz, CDCl₃): δ 0.87 (3H, s, H-19), 0.88 (3H, s, H-18), 3.65 (3H, s,OCH₃), 2.99 (2H, s, CH₂O), 3.32 (3H, s, CH₃O), 4.46 (1H, tt, J₁=11.1,J₂=4.8, H-3), 8.00 (2H, m, H-2′ and H-4′, pyridinium), 8.47 (1H, tt,J₁=7.9, J₂=1.7, H-3′, pyridinium), 8.92-9.06 (2H, m, H-1′ and H-5′,pyridinium). ¹³C NMR (101 MHz, CDCl₃): δ 145.78 (C-1′, C-5′), 142.52(C-3′), 127.27 (C-2′, C-4′), 85.23, 79.75, 59.53, 43.20, 42.47, 40.43,35.22, 34.94, 34.91, 34.63, 33.39, 32.01, 29.34, 28.01, 27.17, 23.47,20.79, 20.51. IR spectrum (CHCl₃): 3140, 3099, 1490, 1024 (pyridinium);1388, 1381 (methyl); 1262, 1254, 1173, 1047 (SO₃); 1109, 1101 (COC). MS(ESI) m/z: 359.3 (100%, M−H-pyridine). HR-MS (ESI) m/z: For C₁₈H₃₁O₅S(100%, M−H-pyridine) calcd: 359.1898; found: 359.1895.

Example 45(3R,5R,8S,9S,10S,13S,14S)-10,13-Dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-3-ol(48)

Metallic zinc (42 g) was under stirring gradually added to a mixture ofcommercially available 3alpha-hydroxy-5beta-androstan-17-one 47 (6 g,0.02 mol) in methanol (90 ml) and dichloromethane (90 ml). The reactionmixture was cooled to 0° C., then, trimethylsilyl chloride (84 ml) wasadded dropwise and the mixture was stirred at room temperature for 14hours. Progress of the reaction was monitored on TLC. The mixture wasfiltered through cellulose and the filtrate was neutralized withsaturated aqueous sodium bicarbonate solution. The product was extractedwith chloroform (3×50 ml), the combined organic phases were washed withaqueous hydrochloric acid (5%, 30 ml), saturated aqueous sodium chloridesolution, dried over anhydrous sodium sulfate and the solvents wereevaporated under reduced pressure. The product was purified by silicagel chromatography (4% acetone in petroleum ether). It was isolated 4.5g (79%) of compound 48: mp 143-144° C., [α]_(D) ²⁰+10.9 (c 0.27, CHCl₃).IR spectrum (CHCl₃): 3608, 3446 (OH); 2972, 2887, 1377, (CH₃); 2935,2865, 1450; (CH₂) 1081, 1065, 1034 (CO). ¹H NMR (400 MHz, CDCl₃): δ 0.68(3H, s, H-18), 0.92 (3H, s, H-19), 3.58-3.67 (1H, m, H-3). ¹³C NMR (101MHz, CDCl₃): 72.05 (C-3), 54.73, 42.28, 41.09, 40.91, 40.64, 39.22,36.65, 36.38, 35.63, 34.89, 30.73, 27.37, 26.99, 25.70, 23.56, 20.99,20.74, 17.65. For C₁₉H₃₂O (276.2) calcd: 82.55% C, 11.67% H; found:82.31% C, 11.82% H.

Example 46 Pyridinium(3R,5R,8S,9S,10S,13S,14S)-10,13-dimethylhexadecahydro-1H-cyclopental[a]phenanthren-3-yl3-sulfate (49)

Compound 49 was prepared according to General Procedure I—Preparation ofC-3 Sulfate from compound 48 (200 mg, 0.72 mmol). White crystals ofsulfate 49 (211 mg, 67%) were isolated: mp 180-182° C. (chloroform),[α]_(D) ²⁰+16.0 (c 0.28, CHCl₃/MeOH, 2:0.1 ml). ¹H NMR (400 MHz, CDCl₃):δ 0.67 (3H, s, H-18), 0.92 (1H, s, CH-19), 4.48 (1H, tt, J₁=11.3,J₂=4.9, H-3), 7.94-8.04 (2H, m, H-2 a H-4, pyridinium), 8.47 (1H, tt,J₁=7.9, J₂=1.6, H-3, pyridinium), 8.97 (2H, dt, J₁=5.6, J₂=1.5, H-1 aH-5, pyridinium). ¹³C NMR (101 MHz, CDCl₃ /CD₃OD): δ 145.57 (C-1′ andC-5′, pyridium), 142.33 (C-3′, pyridinium), 127.07 (C-2″ and C-4′,pyridinium), 79.71 (C-3), 54.67, 42.23, 40.97, 40.75, 40.53, 39.13,36.23, 35.48, 34.67, 33.39, 27.80, 27.10, 26.81, 25.58, 23.35, 20.85,20.61, 17.53. IR spectrum (CHCl₃): 1260, 1178, 1050, 970 (OSO₃). MS (EI)m/z: 355.2 (100%, M-pyridinium). HR-MS (ESI) nilz: For C₁₉H₃₁O₄S caled:355.1949; found: 355.1949.

Example 472-(((3R,5R,8S,9S,10S,13S,14S)-10,13-Dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-3-ypoxy)-2-oxoethanoicacid (50)

5beta-androstan-3alpha-ol 48 (100 mg, 0.362 mmol) was dissolved in drieddichloromethane (2 ml). The solution was cooled to 0° C. andtriethylamine (0.05 ml), a drop of a solution of N,N′-dimethylformamide(0.03 ml) in dichloromethane (2 ml), and oxalic acid chloride (0.09 ml,1.086 mmol) were added while stirring. The mixture was warmed to 10° C.and stirred at this temperature for 2 h. The excess reagent wasdecomposed by addition of water (10 ml) and the solution was stirred atroom temperature for 30 min. Then aqueous layer was separated,dichoromethane evaporated and the residue slurried in ethyl acetate (20ml) and aqueous potassium carbonate (10%, 50 ml). The organic layercontaining the neutral impurities were removed, the aqueous phasecautiously acidified with dilute hydrochloric acid (1N, to pH˜4) andextracted with ethyl acetate (2×25 ml). The combined extracts werewashed with water, dried over anhydrous sodium sulfate and the solventsevaporated under reduced pressure. Crystallization fromacetone/n-heptane yielded 70 mg (55%) of oxalic acid hemiester 50:[α]_(D) ²⁰+27.9 (c 0.32, CHCl₃): ¹H NMR (400 MHz, CDCl₃): δ 0.68 (3H, s,H-18), 0.96 (3H, s, H-19), 4.95 (1H, tt, J₁=11.4, J₂=4.9, H-3).¹³C NMR(101 MHz, CDCl₃): δ 157.83, 157.48, 79.67, 54.69, 42.12, 41.08, 40.83,40.60, 39.13, 36.30, 35.13, 34.88, 31.89, 27.11, 26.81, 26.32, 25.67,23.37, 20.99, 20.72, 17.63. IR spectrum (CHCl₃): 1734, 1726 (C═O), 1268,1246 (C—O). MS (ESI) m/z: 275.3 (100%, M-COCOO), 347.3 (10%, M−H). HR-MS(ESI) in/z: For C₂₁H₃₁O₄ (M−H) caled: 347.2228; found: 347.2232.

Example 482-(((3R,5R,8S,9S,10S,13S,14S)-10,13-Dimethylhexadecahydro-1H-cyclopental[a]phenanthren-3-yl)oxy)-2-oxopropanoicacid (51)

Compound 51 was prepared according to General Procedure III—Preparationof C-3 Hemimalonate from compound 48 (100 mg, 0.36 mmol). Chromatographyon silica gel (1-10% acetone in petroleum ether) afforded 67 mg (51%) ofnon-crystallizing derivative 51: [α]_(D) ²⁰ +27.8 (c 0.35, CHCl₃). ¹HNMR (400 MHz, CDCl₃): δ 0.68 (3H, s, H-18), 0.94 (3H, s, H-19), 3.42(2H, s, H-2′), 4.80-4.90 (1H, m, H-3). ¹³C NMR (101 MHz, CDCl₃): δ169.05, 168.01, 77.04, 54.72, 42.10, 41.10, 40.94, 40.64, 40.20, 39.17,36.33, 35.20, 34.90, 32.16, 27.15, 26.87, 26.59, 25.68, 23.48, 21.02,20.75, 17.66. IR spectrum (CHCl₃): 1755, 1736, 1717 (C═O); 1330 (OH). MS(ESI) m/z: 317.3 (100%, M-HCOOH), 361.3 (20%, M-H), 723.6 (10%, 2M-H).HR-MS (ESI) m/z: For C₂₂H₃₃O₄ (M−H) calcd: 361.2385: found: 361.2380.

Example 49(3R,5R,8S,9S,10S,13S,14S)-10,13-Dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-3-ylN-2-((benzyloxy)-carbonyl)-N-omega-nitro-L-argininate (52)

A mixture of compound 48 (150 mg, 0.54 mmol), the protected Z,NO₂argininate (211 mg, 0.6 mmol) and 4-(N,N-dimethylamino)pyridine (7 mg,0.06 mmol) were dissolved in acetonitrile (20 ml) and dichloromethane(10 ml) and a solution of dicyclohexylcarbodiimide in benzene (1M, 0.813ml) was added. The reaction mixture was stirred under argon for 17 h.The precipitated derivative N,N′-dicyclohexylurea was removed byfiltration and the filtrate poured into saturated sodium bicarbonatesolution, the product was extracted into ethyl acetate (3×25 ml), thecombined organic phases were washed with brine (25 ml) and dried overanhydrous magnesium sulfate. The solvents were evaporated under reducedpressure. Purification of the crude residue (484 mg) by columnchromatography on silica gel (20% acetone in petroleum ether) gave theproduct 52 (226 mg, 68%): mp 132-133° C., [α]_(D) ²⁰−7.5 (c 0.25,CHCl₃). ¹H NMR (500 MHz, CDCl₃): δ 0.69 (3H, s, H-18), 0.95 (3H, s,H-19), 3.22-3.31 (1H, m, 5′b-CH), 3.41-3.50 (1H, m, 5′a-CH), 4.28-4.36(1H, m, 2′-CH), 4.73-4.83 (1H, m, H-3),), 5.12 (2H, s, CH₂-benzyl), 5.75(1H, d, J=8.0, NHCBz), 7.23-7.40 (5H, m, phenyl). ¹³C NMR (101 MHz,CDCl3): δ 171.43, 159.42, 136.01, 128.70, 128.49, 127.88, 76.57, 67.51,54.54, 42.04, 40.98, 40.83, 40.50, 40.41, 40.29, 39.01, 36.24, 35.07,34.78, 33.91, 32.20, 31.17, 27.06, 26.74, 26.58, 25.64, 26.56, 24.95,24.48, 2334, 20.92, 20.63, 17.55. IR spectrum: 1730 (C═O, ester); 1705(C═O, carbamate); 1348, 1326, 1291(NO₂); 1231 (phenyl). MS (ESI) m/z:634 (100%, M+Na), 612 (70%, M+H).

Example 50 (3R,5R,8S,9S,10S,13S,14S)-10,13-Dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-3-yl L-argininate dihydrochloride(53)

To a solution of androstanol-Boc,NO₂-argininate-52 (203 mg, 0.33 mmol)in methanol (9.5 ml) and acetic acid (0.5 ml) was added palladiumcatalyst (Pd/C, 10%, 20.3 mg). The mixture was vigorously stirred undera slight positive pressure of hydrogen for 18 h at room temperature. Thereaction was monitored by TLC, in a mixture of petroleum ether/acetone,1:1. After disappearance of the starting material the catalyst wasfiltered over a small column of celite. The filtrate was diluted withchloroform (5 ml), washed with dilute hydrochloric acid (5%, 5 ml) anddried over anhydrous magnesium sulfate. The precipitated hydrochloride53 (140 mg, 84%) was dried. The product 53 was recrystallized frommethanol/water: mp 238-240° C., [α]_(D) ²⁰+20 (c 0.32, CHCl₃). ¹H NMR(400 MHz, CDCl₃): δ 0.74 (3H, s, H-18), 1.00 (3H, s, H-19), 3.27 (2H, d,J=7.8, H-5′), 4.06 (1H, d, J=7.8, H-2′), 4.89 (1H, m, H-3). ¹³C NMR:(101 MHz, CD₃OD): δ 169.69, 78.49, 62.66, 55.92, 53.69, 43.29, 42.16,42.03, 41.67, 41.54, 37.52, 35.83, 33.19, 28.75, 28.09, 27.92, 27.57,26.52, 25.64, 25.54, 23.75, 21.94, 21.43, 22.13, 17.86. IR spectrum(CHCl₃): 3344, 3164, (NH); 1703, 1681, 1600 (guanidinium); 1727 (C═O,ester). MS (ESI) m/z: 433.4 (100%, M−2HCl). HR-MS (ESI) m/z: ForC₂₅H₄₅N₄O₂ (M−2HCl) calcd: 433.3537; found: 433.3537.

Example 51 (3S,5R,8S,9S,10S,13S,14S)-10,13-Dimethylhexad ecahydro-1H-cyclopenta[a]phenanthren-3-yl 4-methylbenzensulfonate (55)

Compound 55 was prepared according to General Procedure VIII—Tosylationfrom 5beta-androstan-3beta-ol 54 (prepared by the same procedure ascompound 48, 4 g, 14.47 mmol) affording compound 55 (5.88 g, 94%):[α]_(D) ²⁰+2.9 (c 0.61, CHCl₃). ¹H NMR (400 MHz, CDCl₃): δ

0.66 (3H, s, H-18), 0.94 (3H, s, H-19), 2.44 (3H, s, CH₃-tosylate),4.81-4.85 (1H, m, H-3), 7.32 (2H, d, J=8.2, tosylate), 7.79 (2H, d,J=8.2, tosylate). ¹³C NMR (101 MHz, CDCl₃): δ 144.38, 134. 95, 129.83(2×), 127.75 (2×), 81.06, 54.80, 41.08, 40.62, 40.52, 39.19, 36.91,36.10, 34.96, 31.49, 30.25, 26.64, 26.37, 25.96, 25.63, 23.78, 21.77,21.21, 20.71, 17.65. IR spectrum (CHCl₃): 2960 (CH₃); 1174 (SO₂); 905(C—O). MS (ESI) m/z: 883.4 (100%, 2M+Na), 453.2 (80%, M+Na). HR-MS (ESI)m/z: For C₂₆H₃₈O₃NaS (M+Na) calcd: 453.2434; found: 453.2434.

Example 52(3R,5R,8S,9S,10S,13S,14S)-3-Azido-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthrene(56)

Compound 56 was prepared according General Procedure IX—Substitution ofTosylate Protecting Group with Alkali Azide from compound 55 (5.88 g,13.68 mmol). Purification by column chromatography (2% ether inpetroleum ether) afforded 2.8 g (68%) of 3alpha-azide (56) and 0.8 g(19%) of a mixture (1:1) with 3beta-isomer. Compound 56: [α]_(D) ²⁰+22.4(c 0.29, CHCl₃). ¹H NMR (400 MHz, CD₃OD): δ 0.68 (3H, s, H-18), 0.94(3H, s, H-19), 3.31 (1H, tt, J₁=11.8, J₂=4.5, H-3). ¹³C NMR (101 MHz,CDCl₃): δ 61.43, 54.64, 42.57, 41.07, 40.92, 40.61, 39.12, 36.33, 35.85,34.95, 32.63, 27.27, 26.90 (2×), 25.67, 23.63, 20.98, 20.72, 17.65. IRspectrum (CHCl₃): 2937, 2868 (CH₃); 2094 (N₃). MS (ESI) m/z: 274.25(100%, M−N₂H), 259.24 (40%, M−N₃H). HR-MS (ESI) m/z: For C₁₉H₃₁ (M−N₃)calcd: 259.2426; found: 259.2423.

Example 53(3R,5R,8S,9S,10S,13S,14S)-10,13-Dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-3-amine(57)

Compound 57 was prepared according to General Procedure V—CatalyticHydrogenation from compound 56 (2.80 g, 9.29 mmol). Chromatography onsilica gel (20% methanol and 1% triethylamine in dichloromethane)yielded 1.74 g (68%) of amine 57: [α]_(D) ²⁰+14.0 (c 0.47, CHCl₃/MeOH,1.8:0.1). ¹H NMR (400 MHz, CD₃OD): δ 0.67 (3H, s, H-18), 0.93 (3H, s,H-19), 2.80 (1H, m, H-3). ¹³C NMR (101 MHz, CDCl₃): δ 54.64, 51.79,42.69, 41.09, 40.89, 40.64, 39.18, 36.42, 36.22, 34.95, 30.45, 27.38,27.01, 25.72, 25.66, 23.75, 21.00, 20.75, 17.67. IR spectrum(hydrochloride) (KBr): 3104 (NH₃ ^(+);) 2973, 1450, 1377 (CH₃). MS (ESI)m/z: 276.3 (100%, M+H). HR-MS (ESI) m/z: For C₁₉H₃₄N (M+H) calcd:276.2686; found: 276.2686.

Example 54 Ethyl2-(((3R,5R,8S,9S,10S,13S,14S)-10,13-dimethyIhexadecahydro-1H-cyclopenta[a]phenanthren-3-yl)amino)-2-oxoacetate(58)

Compound 58 was prepared according to General Procedure XI—Reaction ofC-3 Amino Group with Ethyl Chlorooxoacetate from compound 57 (100 mg,0.36 mmol) affording 123 mg (90%) of 58: mp 165-167° C.(acetone/n-heptane), [α]_(D) ²⁰+36.7 (c 0.38, CHCl₃). ¹H NMR (400 MHz,CD₃OD): δ 0.68 (3H, s, H-18), 0.95 (3H, s, H-19), 1.38 (3H, t, J=7.1,CH₂CH₃), 3.75-3.87 (1H, m, H-3), 4.34 (2H, q, J=7.1, CH₂CH₃), 6.96 (1H,d, J=8.6, NH). ¹³C NMR (101 MHz, CDCl₃): δ 161.21, 155.77, 63.29, 54.82,50.24, 42.44, 41.10, 41.04, 40.65, 39.22, 35.94, 34.86, 33.26, 29.85,27.57, 27.12, 26.94, 25.68, 23.70, 20.99, 20.73, 17.65, 14.16. IRspectrum (CHCl₃):1696 (C═O); 1377 (OEt). MS (ESI) m/z: 398.3 (100%,M+Na). HR-MS (ESI) m/z: For C₂₃H₃₇O₃NNa (M+Na) calcd: 398.2666; found:398.2668.

Example 552-(((3R,5R,8S,9S,10S,13S,14S)-10,13-Dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-3-yl)amino)-2-oxoaceticacid (59)

To a solution of protected amide 58 (110 mg, 0.29 mmol) in methanol (3ml) cooled to 0° C. was added dropwise a solution of sodium hydroxide(120 mg, 3 mmol) in methanol (2 ml). The reaction mixture was stirred at10° C. for 2 h. It was then poured into water, acidified with dilutehydrochloric acid (5%) to pH-2 and the product extracted into ether(3×10 ml). The combined organic phases were washed with brine, driedover anhydrous sodium sulfate and the solvents evaporated under reducedpressure yielding 59 mg (64%) of the monoamide 59: mp 191-193° C.,[α]_(D) ²⁰+32.4 (c 0.21, CHCl₃/MeOH, 1.8:0.4). ¹H NMR (400 MHz, CD₃OD):δ 0.69 (3H, s, H-18), 0.96 (3H, s, H-19), 3.70-3.85 (1H, m, H-3), 7.14(1H, d, J=8.2, NH). ¹³C NMR (101 MHz, CDCI₃): δ 160.05, 156.62, 54.77,51.29, 42.45, 41.10, 41.04, 40.62, 39.17, 36.32, 35.83, 34.85, 33.08,27.40, 27.08, 26.90, 25.67, 23.67, 21.00, 20.72, 17.65. IR spectrum(sodium salt) (KBr): 1649 (C═O, amide); 1532 (amide); 1377 (CH₃). MS(ESI) mz 346.2 (100%, M−H). HR-MS (ESI) m/z: For C₁₉H₃₂NO₃ (M−H) calcd:346.2388; found 346.2386.

Example 56((3R,5R,8S,9S,10S,13S,14S)-10,13-Dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-3-yl)amin)-3-oxopropanoicacid (61)

Compound 60 was prepared according to General Procedure X—Reaction ofC-3 Amino Group with Methyl 3-Chloro-oxopropionate from compound 57 (100mg, 0.36 mmol) affording methyl ester 60 (130 mg, 96%), which formed (byIR and NMR spectra) equilibrium mixture of keto and enol form, and aftercharacterization by mass spectra was used for the next reaction. MS(ESI) m/z: 398.4 (100%, M+Na), 773.8 (40%, 2M+Na). HR-MS (ESI) m/z: ForC₂₃H₃₇NO₃ Na (M+Na) calcd: 398.2666; found: 398.2666.

A solution of 60 (130 mg, 0.35 mmol) and sodium hydroxide (28 mg, 0.69mmol) in tetrahydrofuran (1.5 ml) and water (1.5 ml) was stirred at roomtemperature for 3 h. The reaction mixture was poured into water andneutral fractions were removed by extraction with ether. The aqueousphase was acidified with dilute hydrochloric acid (5%). The product wasextracted into ethyl acetate (3×15 ml). The combined organic extractswere washed with water and dried over anhydrous magnesium sulfate andthe solvents evaporated under reduced pressure. Chromatography on silicagel (10% acetone in petroleum ether) afforded 48 mg (37%) of 61: mp126-128° C., [α]_(D) ²⁰+25.4 (c 0.25, CHCl₃). ¹H NMR (400 MHz, CD₃OD): δ0.68 (3H, s, H-18), 0.95 (3H, s, H-19), 3.29 (2H, s, COCH₂CO), 3.83 (1H,tdt, J₂=12.4, J₂=8.7, J₃=4.6, H-3), 6.3 (1H, d, J=8.4, NH). ¹³C NMR (101MHz, CDCl₃): δ 168.78, 167.91, 54.83, 50.59, 42.49, 41.09, 41.03, 40.65,39.22, 38.58, 36.31, 35.94, 34.86, 33.33, 27.62, 27.11, 26.94, 25.67,23.68, 20.98, 20.73, 17.64. IR spectrum (KBr): 1731, 1719 (C═O, amide);1630, 1622, 1561 (amide); 1377 (CH₃). MS (ESI) m/z: 360.3 (100%, M−H),721.5 (25%, 2M−H). HR-MS (ESI) m/z: For C₂₂H₃₄NO₃(M−H) calcd: 360.2544;found: 360.2536.

Example 574-(((3R,5R,8S,9S,10S,13S,14S)-10,13-Dimethylhexadecahydro-1H-cyclopenta[a]-phenanthren-3-yl)oxy)-N,N,N-trimethyl-4-oxobutan-1-amoniumchloride (62)

3-Carboxy-N,N,N-trimethylpropane-1-ammonium chloride (69 mg, 0.38 mmol)was suspended in anhydrous CH₂Cl₂ (1 ml) under argon. To the reactionmixture cooled in an ice bath was added dropwise oxalic acid chloride(0.5 ml, 5.82 mmol) followed by a catalytic amount of anhydrousN,N′-dimethylformamide (3 ml, 0.03 mmol). The heterogeneous mixture wasstirred at ambient temperature for 16 h. During this time a clearsolution formed. The liquid portions of the mixture were evaporatedunder reduced pressure and the solid residue was dissolved innitromethane (2 ml) and anhydrous pyridine (0.10 ml, 1.24 mmol) underargon. To this solution was added compound 48 (98 mg, 0.35 mmol). Thereaction was stirred 4 h and then, it was quenched with water (10 ml).The resulting mixture was acidified to pH-4 with aqueous HCI (5%). Theproduct was extracted with chloroform (3×20 ml), the solution washedwith saturated aqueous sodium chloride solution (10 ml), dried overanhydrous magnesium sulfate and evaporated under reduced pressure. Theunreacted starting steroid 48 was removed by trituration with benzeneand the residual product was recrystallized from chloroform/n-heptane(1;1) affording crystals of 62 (122 mg, 79%): mp 225-227° C.(n-heptane/CHCl₃), [α]_(D) ²⁰+24.9 (c 0.23, CHCl₃). ¹H NMR (400 MHz,CDCl₃): δ 0.69 (3H, s, H-18), 0.94 (3H, s, H-19), 2.03-2.14 (2H, br m,H-3′) 2.46-2.50 (2H, br m, H-2′), 3.46 (9H, br s, N(CH₃)₃), 3.63-3.71(2H, br m, H-4′), 4.72 (1H, tt, J₁=11.3, J₂=4.6, H-3). ¹³C NMR (101 MHz,CDCl₃): δ 171.55 (CO), 75.45, 65.73, 54.53, 53.60, 41.98, 40.92, 40.77,40.46, 39.00, 36.18, 35.12, 34.74, 32.26, 30.28, 27.03, 26.71, 26.63,25.51, 23.33, 20.86, 20.57, 18.50, 17.50. IR spectrum (CHCl₃): 2950(CH₃); 1722 (C═O, ester); 1477 (NMe₃ ⁺) 1386 (CH₃); 1230 (NMe₃ ⁺); 1185(CO). MS (ESI) m/z: 404 (100%, M−Cl). (ESI) m/z: For C₂₆H₄₅NO₂ (M−CI)calcd: 404.3523; found: 404.3526.

Example 584-(((3R,5R,8R,9S,10S,13R,14S)-10,13-Dimethyl-2,3,4,5,6,7,8,9,10,11,12,13,14,15-tetra-decahydro-1H-cyclopenta[a]phenanthren-3-yl)oxy)-4-oxobutanoicacid (64)

Compound 64 was prepared according to General Procedure II—Preparationof C-3 Hemisuccinate from compound 63 (103 mg, 0.38 mmol).Chromatography on silica gel (25% ethyl acetate in petroleum ether)afforded 85 mg (60%) of the derivative 64: mp 127.7-128.3° C.(acetone/n-heptane), [α]_(D) ²⁰+30.8 (c 0.27, CHCl₃). ¹H NMR (400 MHz,CDCl₃): δ 0.74 (3H, s, H-18), 0.97 (3H, s, H-19), 2.58-2.71 (4H, m,OCCH₂CH₂CO), 4.75 (1H, tt, J₁=11.3, J₂=4.8, H-3), 5.69 (1H, ddd, J₁=5.6,J₂=2.9, J₃=1.4, H-17), 5.83 (1H, m, H-16). ¹³C NMR (101 MHz, CDCl₃): δ177.03, 171.66, 143.89, 129.29, 74.95, 56.16, 45.64, 42.00, 41.30,36.09, 35.00, 34.92, 34.49, 32.25, 32.01, 29.28, 28.87, 27.00, 26.54,23.31, 20.72, 17.03. IR spectrum (CHCl₃): 1717, 1726 (C═O, COOH); 1578(C═C). MS (ESI) m/z: 397.3 (100%, M+Na). HR-MS (ESI) m/z: For C₂₃H₃₄O₄Na(M+Na) calcd: 397.23493; found: 397.23484.

Example 593-(((3R,5R,8R,9S,10S,13R,14S)-10,13-Dimethyl-2,3,4,5,6,7,8,9,10,11,12,13,14,15-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl)oxy)-3-oxopropanoicacid (65)

Compound 65 was prepared according to General Procedure III—Preparationof C-3 Hemimalonate from compound 63 (102 mg, 0.37 mmol). Chromatographyon silica gel (25% ethyl acetate in petroleum ether) gave (102 mg, 76%)of the derivative 65: mp 156.7-157.8° C. (acetone/n-heptane), [α]_(D)²⁰+30.4 (c 0.31, CHCl₃). ¹H NMR (400 MHz, CDCl₃): δ 0.74 (3H, s, H-18),0.98 (3H, s, H-19), 3.42 (2H, s, H-2′), 4.84 (1H, tt, J₁=11.3, J₂=4.8,H-3), 5.69 (1H, ddd, J₁=5.6, J₂=2.9, J₃=1.4, H-17), 5.83 (1H, m, H-16).¹³C NMR (101 MHz, CDCl₃): δ 169.06, 167.78, 143.84, 129.29, 76.90,56.13, 45.64, 42.00, 41.32, 40.08, 36.06, 34.90 (2×C), 34.48, 32.05,31.99, 26.95, 26.51, 26.38, 23.27, 20.73, 17.03. IR spectrum (CHCl₃):1760, 1735, 1719 (C═O); I587 (C═C). MS (ESI) m/z: 383.2 (100%, M+Na).HR-MS (ESI) m/z: For C₂₂H₃₂O₄Na (M+Na) calcd: 383.2193; found: 383.2192.

Example 60(3R,5R,8R,9S,10S,13S,14S)-10,13-Dimethyl-17-methyIenhexadecahydro-1H-cyclopenta[a]phenanthren-3-ol(66)

Sodium hydride (60% in parafin oil, 80 mg, 1.7 mmol) was added to asolution of methyltriphenylphosphonium bromide (619 mg, 1.73 mmol) indried dimethyl sulphoxide (4 ml) under an inert atmosphere of nitrogenand the reaction mixture was stirred at room temperature for 1 h. Then,a solution of 17-oxo-5beta-androstan-3alpha-ol 47 (100 mg, 0.34 mmol) indried tetrahydrofuran (3 ml) was added and after stirring for 1.5 h at70° C., an aqueous ammonium chloride solution was added. The product wasextracted into chloroform (2×20 ml), the combined organic extracts werewashed with saturated aqueous sodium chloride solution and dried overanhydrous sodium sulfate. The solvents were evaporated under reducedpressure and the residue purified by preparative thin layerchromatography (eluted with 40% ether in petroleum ether) affordingcompound 66 (90 mg, 90%): mp 147-149° C. (acetone/n-heptane), [α]_(D)²⁰+30.5 (c 0.22, CHCl₃). ¹H NMR (400 MHz, CDCl₃): δ 0.75 (3H, s, H-18),0.93 (3H, s, H-19), 2.22 (1H, dtt, J₁=17.7, J₂=8.8, J₃=2, H_(a)-16),2.47 (1H, dddd, J₁=16.9, J₂=10, J₃=4.4, J₄=2.2, H_(b)-16), 3.62 (IH, m,H-3), 4.50-4.62 (2H, m, ═CH₂). ¹³C NMR (101 MHz, CDCl₃): δ 162.07(C-17), 100.79 (C-20), 71.97 (C-3), 54.63, 44.34, 42.32, 40.89, 36.59,36.04, 35.97, 35.57, 34.88, 30.69, 29.60, 27.28, 26.51, 24.32, 23.53,20.86, 18.66. IR spectrum (CHCl₃): 3609, 3451, 1031 (OH); 1653 (C═C). MS(ESI) In/z: 311.3 (100%, M+Na). HR-MS (ESI) m/z: For C₂₀H₃₂ONa (M+Na)calcd: 311.2345, found: 311.2344.

Example 613-(((3R,5R,8R,9S,10S,13S,14S)-10,13-Dimethyl-17-methylenehexadecahydro-1H-cyclo-penta[a]phenanthren-3-yl)oxy)-3-oxopropanoic acid (67)

Compound 67 was prepared according to General Procedure III—Preparationof C-3 Hemimalonate from compound 66 (200 mg, 0.69 mmol). Chromatographyon silica gel (1-10% acetone in petroleum ether) gave compound 67 (239mg, 92%): mp 109-111° C. (acetone/n-heptane), [α]_(D) ²⁰+49.5 (c 0.31,CHCl₃). ¹H NMR (400 MHz, CDCl₃): δ 0.76 (3H, s, H-18), 0.96 (3H, s,H-19), 2.23 (1H, m, H-16a), 2.48 (1H, m, H-16b), 3.40 (2H, s, COCH₂CO),4.62 (2H, m, ═CH₂), 4.83 (1H, m, H-3). ¹³C NMR (101 MHz, CDCl₃): δ169.29 (COOH), 167.84 (COO), 161.96 (C-17), 100.87 (C-20), 76.87 (C-3),54.63, 44.34, 42.14, 40.93, 40.30, 36.01, 35.92, 35.15, 34.90, 32.13,29.61, 27.06, 26.56, 26.38, 24.31, 23.44, 20.91, 18.67. IR spectrum(CHCl₃): 3510 (OH, COOH, monomer); 3120 (OH, COCH, dimer); 3069, 1654,885 (═CH₂); 1778 (C═O, COOH, monomer); 1716 (C═O, COOH, monomer). MS(ESI) m/z: 397.3 (100%, M+Na), 771.6 (10%, 2M+Na). For C₂₃H₃₄O₄ (374.5)calcd: 73.76% C, 9.15% H; found: 72.74% C, 9.37% H.

Example 624(((3R,5R,8R,9S,10S,13S,14S)-10,13-Dimethyl-17-rnethylenhexadecahydro-1H-cyclopenta[a]phenanthren-3-yl)oxy)-4-oxobutanoicacid (68)

Compound 68 was prepared according to General Procedure II—Preparationof C-3 Hemisuccinate from compound 66 (200 mg, 0.69 mmol).Chromatography on silica gel (10% acetone in petroleum ether) gavecompound 68 (265 mg, 99%) as a solid foam: [α]_(D) ²⁰+54.5 (c 0.30,CHCl₃). ¹H NMR (400 MHz, CDCl₃): δ 0.76 (3H, s, H-18), 0.94 (3H, s,H-19), 2.23 (1H, m, H-16a), 2.47 (1H, m, H-16b), 2.56-2.68 (4H, m,OCCH₂CH₂CO), 4.61 (2H, m, ═CH₂), 4.74 (1H, m, H-3). ¹³C NMR (101 MHz,CDCI₃): δ 177.61 (COOH), 171.81 (COO), 162.03 (C-17), 100.82 (C-20),75.09 (C-3), 54.66, 44.35, 42.15, 40.91, 36.04, 35.94, 35.25, 34.91,32.32, 29.62, 29.44, 29.11, 27.11, 26.71, 26.41, 24.32, 23.48, 20.90,18.67. IR spectrum (CHCl₃): 3516 (COOH, monomer), 3100 (COOH, dimer),1754 (C═O, COOH, monomer), 1717 (C═O, COOH, dimer). MS (ESI) m/z: 411.2(100%, M+Na). For C₂₄H₃₆O₄ (388.5) calcd: 74.19% C, 9.34% H; found:73.97% C, 9.38% H.

Example 635-(((3R,5R,8R,9S,10S,13S,14S)-10,13-Dimethyl-17-methylenhexadecahydro-1H-cyclopenta[a]phenanthren-3-yDoxy)-5-oxopentanoicacid (69)

Compound 69 was prepared according to General Procedure IV—Preparationof C-3 Hemiglutarate from compound 66 (200 mg, 0.6 mmol). Chromatographyon silica gel (10% ether in petroleum ether) gave a white solid whichcrystallized from acetone/water gave desired hemiester 69 (134 mg, 53%):mp 84-86° C., [α]_(D) ²⁰+52.3 (c 0.17, CHCl₃). ¹H NMR (400 MHz, CDCI₃):δ 0.76 (3H, s, H-18), 0.95 (3H, s, H-19), 2.16-2.28 (2H, m, H-16a,H-16b), 2.35-2.43 (4H, m, glutaric acid), 4.58-4.65 (2H, m, ═CH₂), 4.74(1H, tt, J₁=11.3, J₂=4.7, H-3). ¹³C NMR (101 MHz, CDCl₃): δ 178.33(COOH), 172.38 (COO), 161.87 (C-17), 100.66 (C-20), 74.48 (C-3), 54.49,44.19, 42.00, 40.75, 35.87, 35.78, 35.11, 34.76, 33.59, 32.90, 32.26,29.46, 26.95, 26.64, 26.25, 24.16, 23.33, 20.74, 19.94, 18.51. IRspectrum (CHCl₃): 3517 (OH, COOH, monomer); 3069, 1654 (═CH₂); 2675 (OH,COOH, dimer); 1756 (C═O, COOH, monomer); 1713 (C═O, COOH, dimer); 1722(C═O, ester). MS (ESI) m/z: 425.2 (100%, M+Na). HR-MS (ESI) m/z: ForC₂₅H₃₈O₄Na (M+Na) calcd: 425.2662; found: 425.2662. For C₂₅H₃₈O₄ (402.6)calcd: 74.59% C, 9.51% H; found: 74.31% C, 9.82% H.

Example 646-(((3R,5R,8R,9S,10S,13S,14S)-10,13-Dimethyl-17-methylenehexadecahydro-1H-cyclopenta[a]phenanthren-3-yl)oxy)-6-oxohexanoicacid (70)

Dicyclohexylcarbodiimide (154 mg, 0.74 mmol) in dried benzene (6 ml) wasadded to a solution of adipic acid (120 mg, 0.82 mmol) in driedtetrahydrofurane (6 ml) under inert atmosphere and the mixture wasstirred at room temperature for 1.5 h. Then, a solution of hydroxyderivative 66 (120 mg, 0.41 mmol) and 4-(N,N-dimethylamino)pyridine (7mg, 0.05 mmol) in dried benzene (7 ml) was added dropwise over 15minutes. The reaction mixture was stirred overnight at room temperature.The solids were filtered off, the solvent evaporated under reducedpressure and the residue purified on silica gel (10% acetone inpetroleum ether) affording hemiester 70 (80 mg, 46%): mp 100-102° C.(acetone/water), [α]_(D) ²⁰+42.5 (c 0.22, CHCl₃). ¹H NMR (400 MHz,CDCl₃): δ 0.76 (3H, s, H-18), 0.95 (3H, s, H-19), 2.18-2.51 (6H, m,H_(a)-16, H_(b)-16, adipic acid), 4.61 (2H, m, ═CH₂), 4.73 (1H, m, H-3).¹³C NMR (101 MHz, CDCl₃): δ 178.35 (COOH), 172.98 (COO), 162.06 (C-17),100.81 (C-20), 74.46 (C-3), 54.65, 44.35, 42.15, 40.90, 36.04, 35.94,35.28, 34.92, 34.45, 33.60, 32.43, 29.62, 27.12, 26.81, 26.42, 24.56,24.32, 24.25, 23.50, 20.90, 18.67. IR spectrum (CHCl₃): 3517 (OH, COOH,monomer); 3069, 1654 (═CH₂); 2675 (OH, COOH, dimer); 1755 (C═O, COOH,monomer); 1712 (C═O, COOH, dimer); 1724 (C═O, ester). MS (ESI) m/z:439.2 (100%, M+Na), 855.5 (5%, 2M+Na). HR-MS (ESI) m/z: For C₂₆H₄₀ONa(M+Na) calcd: 439.2818; found: 439.2817.

Example 65 Ethyl(E)-2-((5R,8R,9S,10S,13S,14S)-10,13-dimethyl-17-oxohexadecahydro-311-cyclopenta[a]phenanthren-3-yliden)-acetateand Ethyl (Z)-2((5R,8R,9S,10S,13S,14S)-10,13-dimethyl-17-oxo hexadecahydro-3H-cyclopenta[a]phenanthren-3-yliden)-acetate (mixture of isomers,72)

Triethyl 2-phosphonoacetate (20.4 ml, 103 mmol) was added dropwise to asuspension of sodium hydride (60% dispersion in parafin oil, 3.9 g, 98mmol) in anhydrous tetrahydrofuran (100 ml) at 0° C. under inertatmosphere. Hydrogen gas released, the heterogeneous reaction mixturebecame clear and was stirred for 30 min. Then,5beta-androstan-3,17-dione 71 (14.9 g, 51.6 mmol) in anhydroustetrahydrofuran (40 ml) was added dropwise. The reaction mixture wasstirred for 2 h at 0° C. under inert atmosphere, then poured intosaturated sodium chloride (200 ml) and the product extracted into ethylacetate (3×150 ml). The combined organic extracts were washed withwater, dried over anhydrous sodium sulfate and the solvents evaporatedunder reduced pressure.

Chromatography of the residue on silica gel (15% ethyl acetate inpetroleum ether) gave an oily mixture of E- and Z - isomers of 72 (1:1ratio by NMR, 18.5 g, 99%): ¹H NMR (400 MHz, CDCl₃): δ 0.85 (6H, s,H-18), 0.95 (6H, s, H-19), 1.23-1.27 (6H, m, OCH₂CH₃), 2.30 (1H, t,J=13.7, H-4α, Z-isomer), 2.40-2.47 (2H, m, H-16), 2.64 (1H, t, J=13.4,H-4α, E-isomer), 3.47 (1H, ddd, J₁=14.5, J₂=3.9, J₃=1.6, H-4β,Z-isomer),3.55-3.61 (1H, m, H-2β), 4.08-4.15 (4H, m, OCH₂CH₃), 5.58 (1H,m, ═CH, E-isomer), 5.60 (1H, m, ═CH, Z-isomer). ¹³C NMR (101 MHz,CDCl₃): δ 221.25, 221.16, 167.05, 166.85, 164.25, 164.05, 113.08,113.05, 59.59, 51.66, 47.99, 45.70, 44.89, 41.15, 41.07, 38.67, 38.29,38.18, 36.04, 35.63, 35.48, 32.80, 31.90, 30.35, 26.99, 26,84, 25.28,24.80, 23.37, 23.25, 21.94, 20.52, 20.45, 14.46, 13.95.

Example 662((3R,5R,8R,9S,10S,13S,14S)-10,13-Dimethyl-17-oxohexadecahydro-1H-cyclopenta[a]phenanthren-3-yl)aceticacid (74)

Substance 73 was prepared according to General Procedure V—CatalyticHydrogenation from compound 72 (18.5 g, 51.6 mmol). The crude oilymixture of 3alpha/3beta-isomer 73 (18.5 g, 99%), which could not becrystallized or partially separated was therefore used for the nextreaction step. A solution of potassium hydroxide (1.8 g, 33 mmol) inwater (5 ml) and ethanol (40 ml) was added to 73 (2 g, 5.54 mmol). Thereaction mixture was heated at 85° C. for 1 h. The progress of thereaction was monitored by thin layer chromatography. After disappearanceof the starting material, the reaction mixture was poured into crushedice and water, acidified with dilute hydrochloric acid (HCl/H₂O, 1:2) topH-1. The product was extracted with chloroform (3×50 ml); the combinedorganic extracts were washed with water, dried over anhydrous sodiumsulfate and the solvents evaporated under reduced pressure. The crudeproduct (1.88 g) by repeated crystallization from ethanol gave pure3α-isomer 74 (133 mg, 7%): mp 130-133° C. (ethanol), [α]_(D) ²⁰+96.5 (c0.25, CHCl₃). ¹H NMR (400 MHz, CDCI₃): δ 0.84 (3H, s, H-18), 0.95 (3H,s, H-19), 2.25 (2H, dd, J₁=7.1, J₂=3.0, CH₂COOH), 2.44 (1H, m, H-16).¹³C NMR (101 MHz, CDCl₃): δ 221.59 (C-17), 177.85 (COOH), 51.69, 48.04,43.33, 41.74, 41.05, 37.12, 36.10, 35.61 (2×C), 35.22, 33.62, 31.92,27.71, 27.14, 25.55, 23.95, 21.98, 20.26, 13.96. IR spectrum (CHCl₃):3518 (COOH, monomer); 3091 (COOH, dimer); 1731 (C═O, COOH, monomer);1706 (C═O, COOH, dimer); 1706 (C═O). MS (ESI) m/z: 355.3 (100%, M+Na),687.6 (20%, 2M+Na). For C₂₁H₃₂O₃ (332.3) calcd: 75.86% C, 9.70% H;found: 75.89% C, 9.57% H.

Example 677-(((3R,5R,8R,9S,10S,13S,14S)-10,13-Dimethyl-17-methylenehexadecahydro-1H-cyclopenta[a]phenanthren-3-yl)oxy)-7-oxoheptanoicacid (75)

Compound 75 was prepared according to the General Procedure VII—WittigReaction Using n-butyl lithium from compound 74 (130 mg, 039 mmol).Chromatography on silica gel (10% ethyl acetate in petroleum ether)recovered starting material 74 (55 mg) and the desired methylenederivative 75 (50 mg, 38%): mp 155-159° C. (acetone), [α]_(D) ²⁰+30.4 (c0.15, CHCl₃). ¹H NMR (400 MHz, CDCl₃): δ 0.76 (3H, s, H-18), 0.95 (3H,s, H-19), 2.17-2.25 (3H, m, H-16a, CH₂COOH), 2.47 (1H, m, H-16b), 4.61(2H, d, J=6.8, ═CH₂). ¹³C NMR (101 MHz, CDCl₃): δ 178.11 (COOH), 162.22(C-17), 100.74 (C-20), 54.72, 44.37, 43.46, 41.83, 41.03, 37.20, 36.09,35.99, 35.68, 35.19, 33.70, 29.64, 27.76, 27.37, 26.53, 24.33, 24.05,20.89, 18.69. IR spectrum (CHCl₃): 3518 (COOH, monomer); 3090 (COOH,dimer); 1733 (C═O, COOH, monomer); 1706 (C═O, COOH, dimer); 3069, 1653(═CH₂). MS (ESI) in/z: 329.2 (100%, M−H), 659.5 (20%, 2M−H). ForC₂₂H₃₄O₂ (330.5) calcd: 79.95% C, 10.37% H; found: 79.66% C, 10.41% H.

Example 682-(((3R,5R,8R,9S,10S,13S,14S)-10,13-Dimethyl-17-methylenehexadecahydro-1H-cyclopenta[a]phenanthren-3-yl)oxy)-N,N,N-trimethyl-2-oxoethan-1-ammoniumchloride (76)

To a solution of betaine (trimethyl glycine, dried overnight at 50° C.,255 mg, 1.6 mmol) in dried dichloromethane was added oxalic acidchloride (2.06 ml, 24 mmol) and N,N′-dimethylformamide (4 drops). Thereaction mixture was stirred at room temperature overnight. Then, thesolvents were evaporated under reduced pressure and the residue wastreated with dried nitromethane (6 ml), dried pyridine (0.3 ml) and thehydroxy derivative 66 (160 mg, 0.55 mmol). The reaction mixture wasstirred at room temperature overnight and then, it was poured into waterand acidified with dilute hydrochloric acid (5%) to pH-4. The productwas extracted into benzene. The benzene extract was dried over anhydrousmagnesium sulfate and the solvent evaporated under reduced pressure.Twofold crystallization (chloroform/n-heptane) afforded quaternaryammonium salt 76 (136 mg, 57%): mp 175-177° C., [α]_(D) ²⁰+43.3 (c 0.24,CHCl₃). ¹H NMR (400 MHz, CD₃OD): δ 0.76 (3H, s, H-18), 0.95 (3H, s,H-19), 3.65 (9H, br s, N(CH₃)₃), 4.62 (2H, d, J₁=7.3, ═CH₂), 4.80 (1H,tt, J₁=11.1, J₂=4.6, H-3), 4.88 (1H, s, H-2′). ¹³C NMR (101 MHz, CDCl₃):δ 164.39, 161.84, 102.93, 100.91, 78.00, 63.48, 54.41, 44.31, 42.18,40.86, 35.90, 35.10, 34.87, 32.16, 29.60, 27.03, 26.57, 26.30, 24.30,23.39, 20.90, 18.68. IR spectrum (CHCl₃): 3070, 1654, 1416 (C═CH₂); 2960(N(CH₃)₃); 1743 (C═O); 1258 (C—O). MS (ESI) m/z: 388.3 (100%, M - Cl),389.3 (30%, M−Cl +1). HR-MS (ESI) m/z: For C₂₅H₄₂NO₂ (M−Cl) calcd:388.3210, found: 388.3209.

Example 69(3R,5R,8S,9S,10S,13R,14S,17S)-10,13,17-Trimethylhexadecahydro-1H-cyclopenta[a]phenanthren-3-ol(77)

Compound 77 was prepared according to General Procedure V—CatalyticHydrogenation from compound 66 (100 mg, 0.34 mmol). Crystallization fromacetone/n-heptane gave methyl derivative 77 (75 mg, 75%): mp 151-153° C.(acetone/n-heptane), [α]_(D) ²⁰+18.1 (c 0.21, CHCl₃). ¹H NMR (400 MHz,CDCl₃): δ 0.52 (3H, s, H-18), 0.82 (3H, d, J=6.8, 17-Me), 0.92 (3H, s,H-19), 3.62 (1H, m, H-3). ¹³C NMR (101 MHz, CDCl₃): δ 72.06 (C-3),56.04, 45.31, 42.41, 42.34, 41.05, 37.91, 36.70, 36.28, 35.66, 34.90,30.75, 30.43, 27.40, 26.81, 24.92, 23.58, 20.76, 13.97, 12.17. IRspectrum (CHCl₃): 3610, 1034 (OH); 1379 (methyl). MS (ESI) m/z: 313.2(100%, M+Na). For C₂₀H₃₄O (290.5) calcd: 82.69% C, 11.80% H; found:82.62% C, 11.37% H.

Example 703-Oxo-3-(((3R,5R,8S,9S,10S,13R,14S,17S)-10,13,17-trimethylhexadecahydro-1H-cyclopenta[a]phenanthren-3-yl)oxy)propanoicacid (78)

Compound 78 was prepared by the General Procedure III—Preparation of C-3Hemimalonate from compound 77 (151 mg, 0.52 mmol). Chromatography onsilica gel (10-20% ethyl acetate in petroleum ether) gave compound 78(162 mg, 82%): mp 158-161.2° C. (acetone/n-heptane), [α]_(D) ²⁰+36.9 (c0.31, CHCl₃). ¹H NMR (400 MHz, CDCI₃): δ 0.53 (3H, s, H-18), 0.82 (3H,d, J=6.8, 17-methyl), 0.95 (3H, s, H-19), 3.36-3.47 (2H, m, OCCH₂CO),4.79-4.89 (1H, m, H-3). ¹³C NMR (101 MHz, CDCI₃): δ 168.80 (COOH),168.08 (COO), 77.05 (C-3), 55.96, 45.32, 42.34, 42.19, 41.04, 40.15,37.84, 36.20, 35.19, 34.18, 32.18, 30.40, 27.16, 26.67, 26.59, 24.88,23.49, 20.77, 13.98, 12.18. IR spectrum (CHCl₃): 3511 (OH, COOH,monomer); 1760 (C═O, COOH, monomer); 1720 (C═O, COOH, dimer); 1386(methyl). MS (ESI) In/z: 399.4(100%, M+Na). HR-MS (ESI) m/z: ForC₂₃H₃₆O₄Na (M+Na) calcd: 399.2506; found: 399.2506.

Example 714-Oxo-4-(((3R,5R,8S,9S,10S,13R,14S,17S)-10,13,17-trimethylhexadecahydro-1H-cyclopenta[a]phenanthren-3-ypoxy)butanoicacid (79)

Compound 79 was prepared according to General Procedure II—Preparationof C-3 Hemisuccinate from compound 77 (200 mg, 0.69 mmol).Chromatography on silica gel (10-20% ethyl acetate in petroleum ether)gave hemiester 79 (123 mg, 89%): mp 141-142° C. (aceton/n-heptane),[α]_(D) ²⁰+34.4 (c 0.27, CHCl₃). ¹H NMR (400 MHz, CDCI₃): δ 0.51 (3H, s,H-18), 0.81 (3H, d, J=6.8, 17-methyl), 0.92 (3H, s, H-19), 2.55-2.66(4H, m, OCCH₂CH₂CO), 4.73 (1H, m, H-3). ¹³C NMR (101 MHz, CDCl₃): δ175.34 (COOH), 172.03 (COO), 74.98 (C-3), 55.94, 45.29, 42.31, 42.17,40.97, 37.83, 36.18, 35.29, 34.89, 32.36, 30.38, 29.65, 29.13, 27.19,26.73, 26.68, 24.87, 23.51, 20.73, 13.97, 12.15. IR spectrum (CHCl₃):3515 (OH, COOH, monomer), 3086 (OH, COOH, dimer), 1755 (C═O, COOH,monomer), 1725 (C═O), 1717 (C═O, COOH, dimer), 1381 (methyl). MS (ESI)m/z: 389.3 (100%, M−H). For C₂₄H₃₈O₄ (390.5) calcd: 73.81% C, 9.81% H;found: 73.76% C, 9.68% H.

Example 725-Oxo-5-(((3R,5R,8S,9S,10S,13R,14S,17S)-10,13,17-trimethylhexadekahydro-1H-cyklopenta[a]phenanthren-3-yl)oxy)pentanoicacid (80)

Compound 80 was prepared according to General Procedure IV—Preparationof C-3 Hemiglutarate from compound 77 (200 mg, 0.69 mmol).Chromatography on silica gel (20-30% ethyl acetate in petroleum ether)gave compound 80 (135 mg, 96%): mp 151-152° C. (acetone/n-heptane),[α]_(D) ²⁰+31.9 (c 0.27, CHCl₃). ¹H NMR (400 MHz, CDCI₃): δ 0.52 (3H, s,H-18), 0.82 (3H, d, J=6.8, 17-methyl), 0.93 (3H, s, H-19), 1.96 (2H, p,J=7.3, H-3′), 2.37 (2H, t, J=7.3, H-2′), 2.43 (2H, t, J=7.3, H-4′), 4.74(1H, m, H-3). ¹³C NMR (101 MHz, CDCl₃): δ 177.67 (COOH), 172.52 (COO),74.74 (C-3), 55.98, 45.32, 42.34, 42.20, 41.01, 37.86, 36.21, 35.32,34.92, 33.77, 32.92, 32.47, 30.41, 27.21, 26.83, 26.70, 24.90, 23.54,20.76, 20.12, 13.99, 12.18. IR spectrum (CHCl₃): 3516 (OH, COOH,monomer); 3089 (OH, COOH, dimer); 1755 (C═O, COOH, monomer); 1723 (C═O);1713 (C═O, COOH, dimer); 1380 (methyl). MS (ESI) m/z: 403.3 (100%, M−H).For C₂₅H₄₀O₄ (404.5) calcd: 74.22% C, 9.97% H; found: 74.08% C, 9.81% H.

Example 73(4S)-4-Amino-5-oxo-5-(((3R,5R,8S,9S,10S,13R,14S,17S)-10,13,17-trimethylhexadecahydro-1H-cyclopenta[a]phenanthren-3-yl)oxy)pentanoicacid (81)

To a stirred mixture of alcohol 77 (500 mg, 1.72 mmol) andBoc-Glu(OBzl)-OH (640 mg, 1.90 mmol) in freshly dried benzene (35 ml)were added 4-(N,N-dimethylamino)pyridine (21 mg, 0.172 mmol) anddicyclohexylcarbodiimide (1M solution in benzene, 2.5 ml) under inertatmosphere at room temperature. After 18 h, the reaction mixture waspoured into saturated sodium bicarbonate solution (40 ml), the productwas extracted into ethyl acetate (3×30 ml), and the combined organicphases were washed with water (2×10 ml). The precipitatedN,N′-dicyclohexylurea was filtered off, the filtrate was dried overanhydrous sodium sulfate and the solvents evaporated under reducedpressure. Further portions of N,N′-dicyclohexylurea was crystallisedfrom ether and filterred off. The filtrate containing the desiredproduct was evaporated under reduced pressure. Chromatography of theresidue on silica gel (10% ethyl acetate in petroleum ether) gave solidwhite foam of protected glutamate (960 mg, 91%). The product wascharacterized by NMR and used crude for the next step. ¹H NMR (400 MHz,CDCl₃): δ 0.53 (3H, s, H-18), 0.83 (3H, d, J=6.8, H-20), 0.94 (3H, s,H-19), 1.43 (9H, s, t-Bu), 2.26-2.43 (2H, m, H-4′), 4.32-4.41 (1H, m,CH-2′), 4.72 (1H, tt, J₁=11.3, J₂=4.7, H-3), 5.17 (2H, d, J=5.1,CH₂-benzyl), 7.34-7.38 (5H, m, phenyl). ¹³C NMR (101 MHz, CDCl₃): δ172.41 (C-1′), 172.37 (C-5′), 155.50 (OCONH), 135.48 (C-1′, phenyl),128.74 (C-3′, C-5′, phenyl), 128.54 (C-4′, phenyl), 128.35 (C-2′, C-6′,phenyl), 80.15 (t-Bu), 74.93 (C-3), 67.32, 55.99, 53.30, 45.32, 42.34,42.19, 41.01, 37.86, 36.21, 35.30, 34.90, 32.38, 30.91, 30.41, 28.46(3×C, t-Bu), 27.87, 27.20, 26.78, 26.69, 24.88, 23.53, 20.76, 13.98,12.17.

To a solution of the protected ester of glutamic acid (950 mg, 1.56mmol) in absolute methanol (55 ml) was added palladium catalyst oncharcoal (10%, 50 mg). The reaction mixture was stirred at roomtemperature under vigorous stirring and a slight positive pressure ofhydrogen for 2 h. The catalyst was filtered off and the solventsevaporated under reduced pressure. The residue was dissolved indichloromethane (8 ml) and stirring was dropwise added trifluoraceticacid (5 ml). The reaction mixture was stirred at room temperature for 30min and then evaporated under reduced pressure to dryness. To theresidue was added a mixture of methanol (10 ml) and pyridine (1 ml) andthe solution was dropwise added into water (60 ml) with crushed ice. Thewhole mixture was kept overnight at 5° C. The solids were collected byfiltration, washed with water and dried to afford the monoester 81 (541mg, 67%, two steps): mp 169-171° C. (metanol), [α]_(D) ²⁰+52.5 (c 0.099,CHCl₃/MeOH, 1.7:0.8). ¹H NMR (400 MHz, CDCl₃/CD₃OD, 6:1): δ 0.48 (3H, s,H-18), 0.78 (3H, d, J=6.7, H-20), 0.90 (3H, s, H-19), 2.42-2.55 (2H, m,H-4′), 3.52-3.61 (1H, m, CH-2′), 4.60-4.72 (1H, m, H-3). ¹³C NMR (101MHz, CDCI₃/CD₃OD, 6:1): δ 173.36 (COOH, COO), 75.22 (C-3), 55.86, 55.83,45.23, 42.25, 42.17, 40.95, 37.76, 36.14, 35.25, 34.83, 32.31, 31.16,3032, 29.76, 27.19, 26.65, 26.57, 24.81, 23.47, 20.70, 13.86, 12.05. IRspectrum (KBr): 2717, 1540, 1491 (NH₃ ⁺); 1745, 1718 (C═O, glutamylester); 1732 (CO); 1635 (COO⁻); 1022 (COC). MS (ESI) m/z: 442.3 (100%,M+Na), 420.4 (45%, M+H). HR-MS (ESI) m/z: For C₂₅H₄₂O₄N (M+H) calcd:

420.3108; found: 420.3108.

Example 743(((3R,5R,8R,9S,10S,13S,14S,Z)-17-Ethylidene-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-3-ol(82)

Hydroxyderivative 82 was prepared according to the literature (Chem.Pharm. Bull., 31, 3819-3828, (1983)).

Example 753-(((3R,5R,8R,9S,10S,13S,14S,Z)-17-Ethylidene-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-3-yl)oxy)-3-oxopropanoicacid (83)

Compound 83 was prepared according to General Procedure III—Preparationof C-3 Hemimalonate from compound 82 (200 mg, 0.66 mmol). Chromatographyon silica gel (1-10% acetone in petroleum ether) gave compound 83 (239mg, 92%): mp 126-128° C. (acetone/petroleum ether), [α]_(D) ²⁰+24.0 (c0.55, CHCl₃). ¹H NMR (400 MHz, CDCl₃): δ 0.86 (3H, s, H-18), 0.95 (3H,s, H-19), 1.64 (3H, dt, J₁=7.1, J₂=2.0, H-21), 3.41 (1H, s, H-2′), 4.84(1H, m, H-3), 5.12 (1H, qt, J₁=7.2, J₂=2.1, H-20). ¹³C NMR (101 MHz,CDCl₃): δ 179.31 (COOH), 171.59 (COO), 150.42 (C-17), 113.45 (C-20),76.94 (C-3), 56.42, 44.59, 41.87, 40.60, 37.50, 35.49, 35.05, 34.80,32.13, 31.64, 29.03, 27.09, 26.58, 26.33, 24.54, 23.39, 21.19, 17.03,13.25. IR spectrum (CHCl₃): 3516 (COOH, monomer), 3114 (COOH, dimer),1751 (C═O, COOH, monomer), 1726 (C═O), 1717 (C═O, COOH, dimer). MS (ESI)rn/z: 425.3 (100%, M+Na). For C₂₅H₃₈O₄ (402.5) calcd: 74.59% C, 9.51% H;found: 74.68% C, 9.42% H.

Example 764-(((3R,5R,8R,9S,10S,13S,14S,Z)-17-Ethylidene-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-3-yl)oxy)-4-oxobuthnoic acid (84)

Compound 84 was prepared according to General Procedure II—Preparationof C-3 Hemisuccinate from compound 82 (200 mg, 0.66 mmol).Chromatography on silica gel (3-20% acetone in petroleum ether) gavehemiester 84 (250 mg, 94%): mp 152-154° C. (acetone/petroleum ether),[α]_(D) ²⁰+58.1 (c 0.21, CHCl₃). ¹H NMR (400 MHz, CDCl₃): δ 0.85 (3H, s,H-18), 0.93 (3H, s, H-19), 1.65 (3H, dt, J₁=7.1, J₂=1.9, H-21),2.57-2.69 (4H, m, OCCH₂CH₂CO), 4.75 (1H, m, H-3), 5.11 (1H, qt, J₁=7.1,J₂=2.0, H-20). ¹³C NMR (101 MHz, CDCl₃): δ 177.07 (COOK 171.83 (COO),150.50 (C-17), 113.40 (C-20), 75.13 (C-3), 56.45, 44.59, 42.07, 40.70,37.54, 35.52, 35.16, 34.82, 32.32, 31.66, 29.46, 29.03, 27.14, 26.74,26.36, 24.56, 23.44, 21.19, 17.03, 13.25. IR spectrum (CHCl₃): 3516(COOH, monomer), 3114 (COOH, dimer), 1751 (C═O, COOH, monomer), 1726(C═O), 1717 (C═O, COOH, dimer). MS (ESI) rn/z: 425.3 (100%, M+Na). ForC₂₅H₃₈O₄ (402.5) calcd: 74.59% C, 9.51% H; found: 74.68% C, 9.42% H.

Example 775-(((3R,5R,8R,9S,10S,13S,14S,Z)-17-Ethylidene-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-3-yl)oxy)-5-oxopentanoicacid (85)

Compound 85 was prepared according to General Procedure IV—preparationof C-3 Hemiglutarate from compound 82 (150 mg, 0.49 mmol).Chromatography on silica gel (5-20% acetone in petroleum ether) gavecompound 85 (178 mg, 86%): mp 100-103° C. (acetone), [α]_(D) ²⁰+57.3 (c0.39, CHCl₃). ¹H NMR (400 MHz, CDCl₃): δ 0.85 (3H, s, H-18), 0.93 (3H,s, H-19), 1.64 (3H, dt, J₁=7.1, J₂=1.9, H-21), 2.34-2.44 (4H, m,hemiglutarate), 4.74 (1H, m, H-3), 5.11 (1H, qt, J₁=7.0, J₂=1.9, H-20).¹³C NMR (101 MHz, CDCl₃): δ 178.19 (COOH), 172.55 (COO), 150.50 (C-17),113.39 (C-20), 74.67 (C-3), 56.44, 44.59, 42.08, 40.69, 37.53, 35.51,35.17, 34.82, 33.77, 33.04, 32.41, 31.66, 27.14, 26.82, 26.36, 24.55,23.44, 21.19, 20.12, 17.03, 13.25. IR spectrum (CHCl₃): 3517 (OH, COOH,monomer), 3116 (COOH, dimer), 1747 (C═O, COOH, monomer), 1722 (C═O),1713 (C═O, COOH, dimer). MS (ESI) rn/z: 439.2 (100%, M+Na), 855.5 (5%,2M+Na). For C₂₆H₄₀O₄ (416.6) calcd: 74.96% C, 9.68% H; found: 74.77% C,9.72% H.

Example 78(3R,5R,8R,9S,10S,13S,14S,17R)-10,13-Dimethyl-17-(prop-1-en-2-yl)hexadecahydro-1H-cyclopentaialphenanthren-3-ol(87)

Compound 87 was prepared according to the General Procedure VII—WittigReaction Using n-Butyl Lithium from 3alpha,5beta-pregnan-20-one 86 (1 g,3.13 mmol). Chromatography on silica gel (10% ethyl acetate in petroleumether) gave compound 87 (760 mg, 76%): mp 149-151° C.(acetone/n-heptane), [α]_(D) ²+16.6 (c 0.39, CHCl₃). ¹H NMR (400 MHz,CDCl₃): δ 0.54 (3H, s, H-18), 0.91 (3H, s, H-19), 1.75 (3H, s, H-21),2.02 (1H, t, J=9.1, H-17), 3.62 (1H, m, H-3), 4.69 (1H, s, H-22a), 4.83(1H, s, H-22b). ¹³C NMR (101 MHz, CDCl₃): δ 145.86 (C-20), 110.78(═CH₂), 71.99 (C-3), 57.57 (C-17), 56.49 (C-14), 43.58, 4231, 40.83,39.25, 36.65, 36.39, 35.56, 34.80, 30.72, 27.34, 26.57, 25.67, 24.78,24.36, 23.54, 21.04, 13.00. IR spectrum (CHCl₃): 3609, 3447, 1033 (OH);3085, 1639 (═CH₂); 1376 (methyl). MS (ESI) rn/z: 339.2 (100%, M+Na). ForC₂₂H₃₆O (316.5) calcd: 83.48% C, 11.46% H; found: 83.49% C, 11.55% H.

Example 793-(((3R,5R,8R,9S,10S,13S,14S,17R)-10,13-Dimethyl-17-(prop-1-en-2-yl)hexadecahydro-4H-cyclopenta[a]phenanthren-3-yl)oxy)-3-oxopropanoicacid (88)

Compound 88 was prepared according to the General ProcedureVIII—Preparation of C-3 Hemimalonate from compound 87 (1 g, 3.13 mmol).Chromatography on silica gel (10% ethyl acetate in petroleum ether) gavecompound 88 (760 mg, 76%): mp 147.7-148.3° C. (acetone/n-heptane),[α]_(D) ²+37.9 (c 0.23, CHCl₃). ¹H NMR (400 MHz, CDCI₃): δ 0.55 (3H, s,H-18), 0.94 (3H, s, H-19), 1.75 (3H, s, H-21), 2.03 (1H, t, J=9.2,H-17), 3.36-3.47 (2H, m, OCCH₂CO), 4.70 (1H, s, ═CH₂), 4.79-4.91 (2H, m,H-3, ═CH₂). ¹³C NMR (101 MHz, CDCl₃): δ 168.86 (COOH), 168.03 (COO),145.84 (C-20), 110.88 (═CH₂), 76.98 (C-3), 57.57 (C-17), 56.44 (C-14),43.59, 42.11, 40.83, 40.20, 39.18, 36.32, 35.11, 34.81, 32.15, 27.11,26.58, 26.44, 25.65, 24.80, 24.33, 23.46, 21.06, 13.02. IR spectrum(CHCl₃): 3599 (OH, COOH, monomer), 3128 (OH, COOH, dimer), 1761 (C═O,COOH, monomer), 1718 (C═O, COOH, dimer), 1638 (═CH₂). MS (ESI) m/z:425.3 (100%, M+Na). HR-MS (ESI) m/z: For C₂₅H₃₈O₄Na (M+Na) calcd:425.2662; found: 420.2700. For C₂₅H₃₈O₄ (402.3) calcd: 74.59% C, 9.51%H; found: 74.32% C, 9.43% H.

Example 804-(((3R,5R,8R,9S,10S,13S,14S,17R)-10,13-Dimethyl-17-(prop-1-en-2-yl)hexadecahydro-1H-cyclopenta [a]phenanthren-3-yl)oxy)-4-oxobutanoic acid (89)

Compound 89 was prepared according to General Procedure II—Preparationof C-3 Hemisuccinate from compound 87 (200 mg, 0.66 mmol).Chromatography on silica gel (20-30% ethyl acetate in petroleum ether)gave compound 89 (323 mg, 96%): mp 148-149.5° C. (acetone/n-heptane),[α]_(D) ²⁰+42.5 (c 0.30, CHCl₃). ¹H NMR (400 MHz, CDCl₃): δ 0.54 (3H, s,H-18), 0.93 (3H, s, H-19), 1.75 (3H, s, H-21), 2.03 (1H, t, J=9.2,H-17), 2.54-2.73 (4H, m, OCCH₂CH₂CO), 4.67-4.79 (2H, m, H-3, ═CH₂), 4.84(1H, s, ═CH₂). ¹³C NMR (101 MHz, CDCl₃): δ 177.32 (COOH), 171.80 (COO),145.84 (C-20), 110.83 (═CH₂), 75.15 (C-3), 57.58 (C-17), 56.46 (C-14),43.60, 42.11, 40.81, 39.20, 36.33, 35.21, 34.82, 32.34, 29.44, 29.06,27.15, 26.73, 26.47, 25.66, 24.81, 24.35, 23.50, 21.05, 13.01. IRspectrum (CHCl₃): 3518 (OH, COOH, monomer), 3100 (OH, COOH, dimer), 1755(C═O, COOH, monomer), 1718 (C═O, COOH, dimer), 1640 (═CH₂). MS (ESI)m/z: 415.2 (100%, M−H). For C₂₆H₄₀O₄ (416.6) calcd: 74.96% C, 9.68% H;found: 74.94% C, 9.65% H.

Example 812((3R,5R,8R,9S,10S,13S,14S,17R)-10,13-Dimethyl-17-(prop-1-en-2-yl)hexadecahydro-1H-cyclopenta[a]fenanthren-3-ypaceticacid (91)

Compound 91 was prepared according to the General Procedure VII—WittigReaction Using n-Butyl Lithium from compound 90 (5beta-pregnan-20-one3-acetatic acid, 202 mg, 0.56 mmol). Chromatography on silica gel (20%ether in petroleum ether) gave compound 91 (80 mg, 40%): mp 198-200° C.(acetone/n-heptane), [α]²⁰ _(D)+27.3 (c 0.17, CHCl₃). ¹H NMR (400 MHz,CDCl₃): δ 0.55 (3H, s, H-18), 0.93 (3H, s, H-19), 2.26 (2H, d, J=7.9,H-2, acetic acid), 4.70 (1H, s, H_(trans), ═CH₂), 4.84 (1H, s, H_(cis),═CH₂). ¹³C NMR (101 MHz, CDCl₃): δ 178.08 (COOH), 145.80 (C-20), 110.60(C-21), 57.44, 56.37, 43.46, 43.25, 41.71, 40.78, 39.11, 36.00, 36.24,35.54, 34.95, 33.55, 27.62, 27.26, 26.44, 25.52, 24.66, 24.21, 23.92,20.89, 12.87. IR spectrum (CHCl₃): 3516 (OH, COOH, monomer); 3085(═CH₂); 2684 (OH, COOH, dimer); 1753 (C═O, COON, monomer); 1706 (COON,dimer). MS (ESI) m/z: 481.2 (100%, M+Na). For C₂₄H₃₈O₂ (358.6) calcd:80.39% C, 10.68% H; found: 80.44% C, 10.79% H.

Example 82 Pyridinium(3R,5R,8R,9S,10S,13S,14S,17S)-17-iodo-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-3-yl3-sulfate (93)

Compound 93 was prepared according to General Procedure I—Preparation ofC-3 Sulfate from compound 92(3alpha-hydroxy-5beta-17beta-iodoandrostane, 255 mg, 0.63 mmol)affording sulfate 93 (298 mg, 72%): mp 118-120° C., [α]_(D) ²⁰+58.5 (c0.39, CHCl₃/MeOH, (1.94:0.20). ¹H NMR (400 MHz, CDCl₃): δ 0.79 (3H, s,H-18), 0.92 (3H, s, H-19), 3.76 (1H, t, J, 9.4, H-17α), 4.39 (1H, tt,J₁=11,0, J₂=5.0, H-3), 7.32 (2H, ddd, J₁=7.6, J₂=4.3, J₃=1.5, H-2′ andH-4′, pyridinium), 7.72 (1H, tt, J₁=7.7, J₂=1.8, H-3′, pyridinium), 8.64(2H, dt, J₁=4.6, J₂=1.7, H-1′ and H-5′, pyridinium). ¹³C NMR (101 MHz,CDCl₃) δ 149.64 (C-1′ and C-5′, pyridinium), 136.45 (C-3′, pyridinium),128.63 (C-2′ and C-4′, pyridinium), 78.88, 50.15, 46.46, 44.29, 42.33,42.12, 40.61, 37.22, 35.55, 34.73, 34.43, 33.45, 27.92, 27.04, 26.47,25.56, 23.19, 20.58, 17.03. IR spectrum (CHCl₃): 3434, 3608 (OH); 1385(CH₃); 1027, 1034 (C—O). MS (ESI) mz: 381.2 (100%, M−H-pyridine). FIR-MS(ESI) m/z: For C₁₉H₃₀O₄IS calcd: 481.0904; found: 481.0908.

Example 83 Pyridinium(3R,5R,8R,9S,10S,13S,14S)-17,17-difluoro-10,13-dimethylhexadeeahydro-1H-cyclopentataiphenanthren-3-yl3-sulfate (95)

Compound 95 was prepared according to General Procedure I—preparation ofC-3 Sulfate from compound 94 (86 mg, 0.28 mmol) affording compound 95(96 mg, 73%): mp 185-187° C., [α]_(D) ²⁰+12.5 (c 0.28, CHCl₃/MeOH,1.97:0.04). ¹H NMR (400 MHz, CDCI₃): δ 0.84 (3H, s, H-19), 0.92 (3H, s,H-19),), 4.46 (1H, tt, J₁=10.8, J₂=5.1, H-3), 8.09-7.99 (2H, m, H-2′ andH-4′, pyridinium), 8.44-8.51 (1H, m, H-3′, pyridinium), 7.96-8.02 (2H,m, H-1′ and H-5′, pyridinium). ¹³C NMR (101 MHz, CDCl₃ and MeOH): δ145.84 (C-1′ and C-5′, pyridinium), 142.42 (C-3′, pyridinium), 127.27(C-2′ and C-4′, pyridinium), 79.51, 58.64, 49.62, 42.23, 40.48, 35.72,35.45, 34.70, 33.44, 33.09, 29.23, 27.87, 26.89, 25.69, 23.34, 22.44,20.06, 18.56, 13.46. IR spectrum (CHCl₃): 1350, 1121 (CF₂); 1247, 1168,973, 947 (OSO₃). MS (ESI) m/z: 391.2 (100%, M−1-pyridine). HR-MS (ESI)m/z: For C₁₉H₂₉F₂S calcd 391.1760; found 391.1757.

Example 84(3R,5R,8R,9S,10S,13S,14S,17S)-10,13-Dimethylhexadecahydrospiro[cyclopenta[a]phenanthren-17,2′-oxiran]-3-ol(96)

Potassium tert-butoxide (267 mg, 2.38 mmol) and trimethylsulfoniumiodide (486 mg, 2.38 mmol) were added all at once to a solution5beta-androstan-3alpha-ol (47, 346 mg, 1.19 mmol) in driedN,N-dimethylformamide (6 ml) under inert atmosphere. The reactionmixture was stirred at room temperature overnight. Then, a saturatedaqueous sodium chloride solution was added and the product was extractedwith chloroform (3×30 ml). The combined organic extracts were dried overanhydrous sodium sulfate and the solvents evaporated under reducedpressure. The residue was purified by column chromatography (0-15% ethylacetate in petroleum ether) giving compound 96 (284 mg, 78%): mp 149-150° C. (ether/petroleum ether). ¹H NMR (400 MHz, CDCl₃): δ 0.85 (3H, s,H-18), 0.92 (3H, s, H-19), 2.60 (1H, d, J=5.1, H-20a), 2.89 (1H, d,J=5.1, H-20b), 3.63 (1H, m, H-3). ¹³C NMR (101 MHz, CDCl₃): δ 71.84(C-3), 70.71 (C-17), 53.72 (C-20), 53.05 (C-14), 42.23, 40.79, 40.38,36.55, 36.15, 35.53, 34.84, 34.32, 30.66, 29.28, 27.18, 26.08, 23.70,23.45, 20.34, 14.48. HR-MS (ESI) m/z: For C₂₀H₃₂O₂Na (M+Na) calcd:327.2294; found: 327.2293.

Example 85 Pyridinium(3R,5R,8R,9S,10S,13S,14S,17S)-10,13-dimethylhexadecahydrospiro-[cyclopenta[a]phenanthren-17,2′-oxiran]-3-yl3-sulfate(97)

Compound 97 was prepared according to General Procedure I—Preparation ofC-3 Sulfate from compound 96 (50 mg, 0.16 mmol) affording compound 97(36 mg, 47%) as a foam: [α]_(D) ²⁰+8.5 (c 0.14, CHCl₃). ¹H NMR (400 MHz,CDCI₃): δ 0.83 (3H, s, H-18), 0.91 (3H, s, H-19), 2.64 (1H, d, J₃=9.3,H-20a), 3.40 (1H, d, J=10.5, H-20b), 4.43 (1H, m, H-3), 7.57 (2H, ddd,J₁=7.6, J₂=6.1, J₃=1.4, H-2′, H-4′, pyridinium), 7.99 (1H, tt, J₁=7.7,J₂=1.8, H-3′, pyridinium), 8.75 (2H, d, J=4.6, H-1′, H-5′, pyridinium).¹³C NMR (101. MHz, CDCI₃): δ 83.60 (C-17), 71.83 (C-3), 58.54 (CH₂N₃),51.52, 46.19, 42.12, 40.64, 36.69, 36.50, 35.49, 34.99, 34.80, 32.35,30.62, 27.15, 2634, 23.72, 23.43, 20.50, 14.30. IR spectrum (CHCl₃):3140, 3093; 1638 (pyridinium); 1252, 1147 (SO₃). MS (ESI) m/z: 383.3(40%, M−H-pyridine). HR-MS (ESI) m/z: For C₂₀H₃₁O₅S(M−H-pyridine) calcd:383.1897; found: 383.1895.

Example 86(3R,5R,8R,9S,10S,13S,14S,17S)-17-(Azidomethyl)-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-3,17-diol(98)

A solution of compound 96 (560 mg, 1.83 mmol), sodium azide (341 mg,5.68 mmol) and ammonium chloride (341 mg, 6.37 mmol) in ethanol (28 ml)and water (5.6 ml) was heated at 90° C. overnight. Water was then added,the ethanol was evaporated and the product extracted with chloroform(2×50 ml). The combined organic extracts were dried over anhydroussodium sulfate and the solvents evaporated under reduced pressure. Theoily residue by column chromatography on silica gel (30% ether inpetroleum ether) gave 500 mg (78%) of a white solid foam 98: [α]_(D)²⁰0.0 (c 0.11, CHCl₃). ¹H NMR (400 MHz, CDCl₃): δ 0.86 (3H, s, H-18),0.93 (3H, s, H-19), 3.26 (1H, d, J=12, H_(a)—CH₂N₃), 3.54 (1H, d, J=12,H_(b)—CH₂N₃), 3.63 (1H, m, H-3). ¹³C NMR (101 MHz, CDCl₃): δ 83.60(C-17), 71.83 (C-3), 58.54 (CH₂N₃), 51.52, 46.19, ¹³C NMR (100 MHz,CDCl₃): δ 71.84 (C-3), 70.71 (C-17), 53.72 (C-20), 53.05 (C-14), 42.23,40.79, 40.38, 36.55, 36.15, 35.53, 34.84, 34.32, 30.66, 29.28, 27.18,26.08, 23.70, 23.45, 20.34, 14.48. IR spectrum (CHCl₃): 3613, 1037(3α-OH); 3562, 1116 (17β-OH); 2106 (azid). MS ESI mz: 370.2 (100%,M+Na). HR-MS (ESI) m/z: For C₂₀H₃₃O₂N₃Na (M+Na) calcd: 370.2465; found:370.2464.

Example 87(4bR,6aR,8R,10aS,10bS,12aS)-8-Hydroxy-10a,12a-dimethylhexadeeahydrochrysen-1(2H)-one(99)

Sodium iodide (948 mg, 6.3 mmol) was added to a solution of compound 98(220 mg, 0.63 mmol) in dried acetonitrile (10 ml). Trimethylsilylchloride (0.8 ml, 26.6 mmol) was then added dropwise under inertatmosphere. The reaction mixture was stirred at room temperature and theprogress of the reaction monitored by TLC. After complete conversion,dilute hydrochloric acid (5%, 10 ml) was added. The product wasextracted with chloroform (2×40 ml). The combined organic extracts werewashed with sodium sulfite solution, saturated aqueous sodium chloridesolution, dried over anhydrous sodium sulfate and the solventsevaporated under reduced pressure. Yellowed residue was chromatographedon silica gel (30% ether in petroleum ether) gave 140 mg (73%) of 99: mp201-201° C. (ether/petroleum ether), [α]_(D) ²⁰−23.6 (c 0.12, CHCl₃). ¹HNMR (400 MHz, CDCI₃): δ 0.91 (3H, s, H-18), 1.06 (3H, s, H-19), 2.04(1H, m, H-16a), 2.19 (1H, m, H-17a), 2.61 (1H, td, J₁=14.0 J₂=6.8,H-17b), 3.61 (1H, m, H-3). ¹³C NMR (101 MHz, CDCl₃): δ 216.70 (C-17a),71.86 (C-3), 51.73, 48.53, 41.71, 39.88, 37.34, 36.36, 35.68, 35.09,35.00, 32.79, 30.74, 27.27, 26.11, 25.93, 23.49, 23.16, 19.87, 17.06. IRspectrum (CHCl₃): 3609, 1037 (OH); 1698 (C═O). MS ESI m/z: 327.2 (100%,M+Na), 631.5 (15%, 2M+Na). For C₂₀H₃₂O₂ (304.4) calcd: 78.90% C, 10.59%H; found: 78.55% C, 10.49% H.

Example 88(2R,4aS,4bS,6aS,10bS,6aS,12aR)-4a,6a-Dimethylolitadecahydrochrysen-2-ol(100)

Compound 100 was prepared from compound 99 (110 mg, 0.36 mmol)analogously to the preparation of compound 48. Chromatography of theresidue on silica gel (30% ether in petroleum ether) gave 100 (68 mg,65%): mp 187-189° C. (acetone/n-heptane), [α]_(D) ²⁰+17.1 (c 020,CHCl₃). ¹H NMR (400 MHz, CDCl₃): δ 0.78 (3H, s, H-18), 0.89 (3H, s,H-19), 3.62 (1H, m, H-3). ¹³C NMR (101 MHz, CDCI₃): δ 72.07 (C-3),51.26, 42.54, 42.45, 42.00, 40.77, 36.52, 36.03, 35.20, 35.09, 33.77,30.77, 27.45, 27.39, 25.53, 24.23, 23.62, 21.66, 20.38, 17.09. IRspectrum (CHCl₃): 3609, 3447, 1031 (OH). MS ESI m/z: 313.3 (100%, M+Na).For C₂₀H₃₄O (290.4) calcd: 82.69% C, 11.80% H; found: 82.42% C, 11.71%H.

Example 89 Pyridinium(2R,4aS,4bS,6aS,10bS,6aS,12aR)-4a,6a-dimethyloetadecahydrochrysen-2-yl2-sulfate (101)

Compound 101 was prepared according to General Procedure I—Preparationof C-3 Sulfate from compound 100 (30 mg, 0.10 mmol) affording sulfate101 (43 mg, 93%): mp 173-174° C., [α]_(D) ²⁰+20.4 (c 0.27, CHCl₃).¹H NMR(400 MHz, CDCI₃): δ 0.76 (3H, s, H-18), 0.87 (3H, s, H-19), 4.45 (1H, m,H-3), 8.00 (2H, m, H-2′, H-4′, pyridinium), 8.48 (1H, t, J=7.8, H-3′,pyridinium), 8.98 (2H, d, J=5.5, H-1 ′, H-5′, pyridinium). ¹³C NMR (101MHz, CDCI₃): δ 145.87 (C-1′, C-5′), 142.38 (C-3′), 127.32 (C-2′, C-4′),79.82 (C-3), 51.28, 42.52, 42.45, 42.02, 40.67, 35.95, 35.13, 34.97,33.75, 33.34, 27.92, 27.3, 27.22, 25.45, 24.20, 23.53, 21.63, 20.34,17.07. IR (CHCl₃): 3139, 3100, 1637, 1490 (pyridinium); 1263, 1238,1235, 1043 (SO₃). MS ESI m/z: 369.3 (100%, M−H-pyridine). HR-MS (ESI)m/z: For C₂₀H₃₃O⁴S (100%, M−H-pyridine) calcd: 369.2105; found:369.2103.

Example 903-(((2R,4aS,4bS,6aS,10bS,12aR)-4a,6a-Dimethyloctadecallydrochrysen-2-yl)oxy)-3-oxopropanoicacid (102)

Compound 102 was prepared according to General Procedure III—Preparationof C-3 Hemimalonate from compound 100 (132 mg, 0.45 mmol).Chromatography on silica gel (30% ethyl acetate in petroleum ether) gavecompound 102 (157 mg, 92%): mp 145.3-147.2° C. (acetone/n-heptane),[α]²⁰ _(D)+25.3 (c 0.29, CHCl₃). ¹H NMR (400 MHz, CDCl₃): δ 0.79 (3H, s,H-18), 0.91 (3H, s, H-19), 3.36-3.47 (2H, m, OCCH₂CO), 4.80-4.90 (1H, m,H-3). ¹³C NMR (101 MHz, CDCl₃): δ 168.89 (COOH), 168.08 (COO), 77.05(C-3), 51.19, 42.51, 42.35, 41.79, 40.75, 40.17, 35.95, 35.05, 34.73,33.77, 31.99, 27.43, 27.15, 26.58, 25.39, 24.20, 23.53, 21.62, 20.39,17.08. IR spectrum (CHCl₃): 3510 (OH, COOH, monomer); 3131 (OH, COOH,dimer); 1759 (C═O, COOH, monomer); 1734 (C═O); 1717 (C═O, COOH, dimer);1410, 1291 (C—O, COOH, dimer); 1345, 1178 (C—O, COOH, monomer). MS (ESI)m/z: 399.3 (100%, M+Na). (ESI) m/z: For C₂₃H₃₆O₄Na (M+Na) calcd:399.2506, found: 399.2506.

Example 914-(((2R,4aS,4bS,6aS,10bS,12aR)-4a,6a-Dimethyloctadecahydrochrysen-2-yl)oxy)-4-oxobutanoicacid (103)

Compound 103 was prepared according to General Procedure II—PreparationC-3 Hemisuccinate from compound 100 (100 mg, 0.34 mmol). Chromatographyon silica gel (10% ether in petroleum ether) gave compound 103 (81 mg,60%): mp 136-138° C. (acetone/water), [α]_(D) ²⁰+34.8 (c 0.21, CHCl₃).¹H NMR (400 MHz, CDCl₃): δ 0.79 (3H, s, H-18), 0.91 (3H, s, H-19), 2.59(2H, ddd, J₁=7.6, J₂=6.1, J₃=1.4, succinic acid) 2.68 (2H, ddd, J₁=7.3,J₂=6.1, J₃=1.3, succininic acid), 4,75 (1H, tt, J₁=11.3, J₂=4.7, H-3).¹³C NMR (100 MHz, CDCl₃): δ 177.29 (COOH), 171.66 (COO), 75.05 (C-3),51.05, 42.37, 42.23, 41.64, 40.58, 35.81, 34.93, 34.68, 33.62, 32.03,29.28, 28.92, 27.29, 27.04, 26.58, 25.26, 24.05, 23.41, 21.48, 20.23,16.93. IR spectrum (CHCl₃): 3517 (OH, COOH, monomer); 2674 (OH, COOH,dimer); 1753 (C═O, COOH, monomer); 1717 (C═O, COOH, dimer); 1727 (C═O,ester). MS (ESI) m/z: 413.2 (100%, M+Na). HR-MS (ESI) m/z: For C₂₄H₃₉O₄(M+H) calcd: 391.2843; found: 391.2839. For C₂₄H₃₈O₄ (402.6) calcd:73.81% C, 9.81% H; found: 73.54% C, 9.88% H.

Example 925-(((2R,4aS,4bS,6aS,10bS,12aR)-4a,6a-Dimethyloktadecahydrochrysen-2-yl)oxy)-5-oxopentanoicacid (104)

Compound 104 was prepared according to General Procedure IV—Preparationof C-3 Hemiglutarate from compound 100 (106 mg, 0.36 mmol).Chromatography on silica gel (1-10% acetone in petroleum ether) gavecompound 104 (137 mg, 93%): mp 148.7-151.0° C. (acetone/n-heptane),[α]_(D) ²⁰+31.7 (c 0.33, CHCl₃). ¹H NMR (400 MHz, CDCl₃): δ 0.78 (3H, s,H-18), 0.90 (3H, s, H-19), 1.96 (2H, p, J=7.3, H-3′), 2.36 (2H, t,J=7.3, H-2′), 2.43 (2H, t, J=7.3, H-4′), 4.74 (1H, m, H-3). ¹³ C NMR(101 MHz, CDCI₃): δ 177.85 (COOH), 172.54 (COO), 74.74 (C-3), 51.22,42.54, 42.39, 41.81, 40.74, 35.98, 35.11, 34.87, 33.78 (2×C), 32.99,32.29, 27.45, 27.21, 26.84, 25.43, 24.21, 23.58, 21.65, 20.39, 20.14,17.09. IR spectrum (CHCl₃): 3517 (OH, COOH, monomer); 3087 (OH, COOH,dimer); 1750 (C═O, COOH, monomer); 1720 (C═O), 1714 (C═O, COOH, dimer);1381 (CH₃). MS (ESI) m/z: 427.3 (100%, M+Na). HR-MS (ESI) m/z: ForC₂₅H₄₀O₄Na (M+Na) calcd: 427.2819; found: 427.2819.

Example 93 5-Benzyl14((2R,4aS,4bS,6aS,10bS,12aR)-4a,6a-dimethyloctadecahydrochrysen-2-yl)-N-(tert-butoxycarbonyl)-L-glutamate(105)

A solution of dicyclohexylcarbodiimide (1M, benzene, 1 ml) was added toa stirred solution of compound 100 (200 mg, 0.68 mmol),4-(N,N-dimethylamino)pyridine (8.5 mg, 0.068 mmol) and5-benzyl-N-benzyloxycarbonyl-L-glutamic acid (Boc-Glu(OBzl)-OH, 255 mg,0.75 mmol) in dried benzene (15 ml) and the reaction mixture was stirredat room temperature under inert atmosphere for 5 h. Then, the solidswere filtered off and washed with dry benzene. The reaction mixture wasconcentrated (about ⅔ of volume), saturated aqueous sodium bicarbonatesolution was added and the product extracted into chloroform (2×50 ml).The combined organic extracts were washed with saturated aqueous sodiumchloride solution and dried over anhydrous sodium sulfate.Chromatography on silica gel (3% acetone in petroleum ether) gave oilycompound 105 (375 mg, 89%): [α]_(d) ²⁰+17.5 (c 0.19, CHCl₃). ¹H NMR (400MHz, CDCI₃): δ 0.78 (3H, s, H-18), 0.90 (3H, s, H-19), 1.43 (9H, s,t-butyl), 2.15-2.25 (1H, m, H-3′), 2.37-2.52 (2H, m, H-4′), 4.28 (1H, m,H-2′), 4.76 (1H, m, H-3), 5.10 (3H, m, NH, OCH₂Ph), 7.35 (5H, m,OCH₂Ph). ¹³C NMR (101 MHz, CDCl₃): δ 172.77 (C-1′, C-5′), 156.15 (NHCO),135.97 (C-1′, benzyl), 128.72 (C-2″, C-6″, benzyl), 128.42 (C-4″,benzyl), 128.35 (C-3″, C-5″, benzyl), 80.06 (OCMe₃), 76.04 (C-3), 66.62(OCH₂Ph), 53.23, 51.15, 42.51, 42.34, 41.81, 40.72, 35.97, 35.08, 34.77,33.78, 32.15, 30.49, 28.48 (3×C, OCMe ₃), 28.16, 27.44, 27.17, 26.72,25.39, 24.21, 23.54, 21.65, 20.40, 17.09. IR spectrum (CHCl₃): 3436,1713 (NH-Boc); 3092, 3068, 1454 (benzyl); 1730 (C═O); 1467, 1368, 1381,1392 (Boc). MS (ES!) m/z: 632.4 (100%, M+Na), 1242.6 (5%, 2M+1). ForC₃₇H₅₅NO₆ (609.8) calcd: 72.87% C, 9.09% H, 2.30 % N; found: 73.15% C,9.22% H, 2.15%N.

Example 94(4S)-4-Amino-5-(((2R,4aS,4bS,6aS,10bS,12aR)-4a,6a-dimethyloctadekahydrochrysen-2-yl)oxy)-5-oxopentanoicacid (106)

A solution of the protected steroid derivative 105 (118 mg, 0.19 mmol)in methanol (5 ml) was stirred in the presence of a palladium catalyston activated carbon (Pd/C, 10%, 5 mg) under slight positive pressure ofhydrogen at room temperature. The reaction was followed by thin layerchromatography (petroleum ether/acetone, 1:1). After 1.5 h, the catalystwas filtered off on a short column of silica gel and washed withchloroform. The combined organic fractions were concentrated.Chromatography on silica gel (10% acetone in petroleum ether) gavetert-butyloxycarbonyl-protected product (62 mg, 62%). The oily residuewas dissolved in concentrated trifluoroacetic acid (1 ml) and thesolution was allowed to stand at room temperature for 15 min.Hydrochloric acid was then removed by blowing a stream of nitrogen. Tothe residue was added a mixture of pyridine/methanol (0.5:4.5 mL) andthe resulting mixture was added dropwise to a mixture of ice and water(5 ml). After 5 h, the white precipitate was filtered off and dried at50° C. overnight affording compound 106 (34 mg, 68%): mp 162 - 165° C.(methanol), [α]_(D) ²⁰+20.7 (c 0.26, CHCl₃/MeOH, 1.80:0.04). ¹H NMR (400MHz, CDCl_(3/)MeOH): δ 0.72 (3H, s, H-18), 0.85 (3H, s, H-19), 2.41 (2H,m, H-3′, H-4′), 3.68 (1H, m, H-2′), 4.75 (1H, m, H-3). ¹³C NMR (101 MHz,CDCl₃/MeOH): δ 177.55 (COOH), 171.27 (COO), 76.71 (C-3), 53.42, 50.95,42.22, 42.06, 41.59, 40.54, 35.72, 34.82, 34.43, 33.49, 33.27, 31.83,27.47, 27.14, 26.92, 26.42, 25.15, 23.94, 23.18, 21.33, 20.12, 16.73. IRspectrum (CHCl₃): 2650, 2170, 1610 (NH₃ ⁺); 1743 (C═O); 1571 (COO⁻). MS(ESI) m/z: 418.3 (100%, M−H), 837.5 (40%, 2M−H). HR-MS (ESI) m/z: ForC₂₅H₄₀NO₄ (M−H) calcd: 418.2962; found: 418.2959.

Example 951-((3R,5R,8R,9S,10S,13S,14S)-3-(Methoxymethoxy)-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17(2H,10H,14H)ylidene)-2-(tosyloxy)hydrazine(108)

To a solution of 3alpha-methoxy-5beta-andostan-17-one 107 (1.00 g, 3mmol) and tosyl hydrazide (1.63 g, 8.5 mmol) in dried methanol (70 ml)was added powdered molecular sieve (40 msh, 2 g) The mixture wasrefluxed while stirring under inert atmosphere for 72 h. The progress ofthe reaction was monitored by TLC, using petroleum ether/acetone, 4:1.The reaction mixture was cooled to room temperature and filtered off.The filtrate was concentrated under reduced pressure. The residue wasdissolved in toluene (100 ml), the precipitate was filtered off and thefiltrate was evaporated under reduced pressure. The residue wasdissolved in ethyl acetate. The solution was washed with saturatedaqueous sodium bicarbonate (3×25 ml), brine (25 ml) and dried overanhydrous sodium sulfate. The solvents were evaporated under reducedpressure and the residue purified by column chromatography on silica gel(15% ethyl acetate in petroleum ether) affording 1.05 g of hydrazone 108(67.4%): mp 97.4-99.5° C., [α]_(D) ²⁰+44.2 (c 0.33, CHCl₃). ¹H NMR (400MHz, CDCl₃): δ 0.82 (3H, s, H-18), 0.92 (3H, s, H-19), 2.43 (3H, s,Tos), 3.36 (3H, s, H-2′, MOM), 3.52 (1H, m, H-3), 4.67 (2H, s, H-1′,MOM) 7.29 (2H, m, H-2″ and H-4″, Tos), 7.81 (2H, d, J=8.3, H-1″ andH-5″, Tos). ¹³C NMR (101 MHz, CDCI₃): δ 129.49, 128.64, 128.02, 125.96,94.62, 55.16, 53.29, 41.95, 40.58, 35.32, 35.23, 34.85, 33.53, 31.76,27.69, 26.91, 25.83, 25.34, 23.25, 21.82, 21.64, 20.15, 20.09, 19.79,16.65, 13.69. IR spectrum (CHCl₃): 2938 (CH₂); 1659 (C═N); 1167 (SO₃);1045, 1036 (COCOC). MS (ESI) m/z: 503.2 (10%, M−CH₃). For C₂₈ H₄₂N₂O₅S(518.7) calcd: 64.84% C, 8.16% H, 5.40% N; found: 65.17% C, 8.50% H,5.08% N.

Example 96(3R,5R,8R,9S,10S,13R,14S)-3-(methoxymethoxy)-10,13-dimethyl-2,3,4,5,6,7,8,9,10,11,12,13,14,15-tetradeeahydro-1H-cyclopentafphenanthrene (109)

Tosylhydrazone 108 (1 g, 1.93 mmol) in dried tetrahydrofuran (30 ml) wascooled to 0° C. and while stirring, methylithium (14 ml, 1.6 M ether)was added under inert atmosphere. The reaction was stirred 2 h at 0° C.overnight at room temperature. The mixture was then recooled to 0° C.and quenched with water (50 ml). The product was extracted into ethylacetate (3×25 ml), the combined organic extracts washed with aqueouscitric acid (5%, 30 ml), saturated aqueous sodium chloride solution anddried over anhydrous sodium sulfate. The solvents were evaporated underreduced pressure. The residue (880 mg) was purified by columnchromatography on silica gel (1% acetone in petroleum ether) yielding606.4 mg (98%) of non-crystallising olefin 109: [α]_(D) ²⁰+20.2 (c 0.35,CHCl₃). ¹H NMR (400 MHz, CDCl₃): δ 0.74 (3H, s, H-18), 0.95 (3H, s,H-19), 3.37 (3H, s, H-2′ MOM), 3.53 (1H, tt, J₁=11.2, J₂=4.7, H-3), 4.69(2H, s, H-1′, MOM), 5.67 (1H, ddd, J₁=5.8, J₂=3.0, J₃=1.5, H-16), 5.83(1H, ddd, J₁=5.8, J₂=2.5, J₃=1.1, H-16). ¹³C NMR (101 MHz, CDCl₃): δ144.08 (C-17), 129.45 (C-16), 94.75 (O—C—O), 77.01 (C-3), 56.33, 55.31,45.81, 42.44, 41.44, 36.27, 35.52, 35.21, 34.68, 33.82, 32.19, 27.84,27.39, 26.76, 23.56, 20.86, 17.19. IR spectrum (CHCl₃): 3049 (C═C); 2935(CH₂); 1047, 1036 (COCOC). MS (ESI) m/z: 341.4 (100%, M+Na). HR-MS (ESI)m/z: For C₂₁H₃₄O₂Na (M+Na) calcd: 341.2451; found: 341.2452. ForC₂₁H₃₄O₂(318.5) calcd: 79.19% C, 10.76% H; found: 79.29% C, 10.85% H.

Example 97(3R,5R,8S,9S,10S,13R,14S,16R)-3-(Methoxymethoxy)-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-16-oI(110)

To a saturated solution of the dimer of 9-borabicyclo[3.3.1]nonane (102ml, 51 mmol) which was cooled to 0° C. under inert atmosphere was addeda solution of olefin 109 (2 g, 6.30 mmol) in tetrahydrofuran (42 ml).The reaction mixture was stirred at 0° C. under argon for 4 h. Water wasadded (32.6 ml) and aqueous sodium hydroxide (10%, 32.6 ml), hydrogenperoxide (30%, 48.6 ml), and the mixture was stirred at room temperatureovernight. Then, sodium sulfite (3.36 g), acetic acid (98%, 16.4 ml),water (80.8 ml) and aqueous citric acid (5%, 81.8 ml) were added. Theproduct was extracted into ethyl acetate (3×80 ml). The combined organicextracts were washed with saturated aqueous sodium chloride solution (50ml), aqueous sodium bicarbonate (5%, 2×50 ml), again with saturatedaqueous sodium chloride solution (50 ml) and dried over anhydrous sodiumsulfate. The organic phase was evaporated under reduced pressure andchromatography of the residue on silica gel (1-5% acetone in petroleumether) gave 1.26 mg (60%) of hydroxy derivative 110: mp 98.7-100° C.(actone/n-heptane), [α]_(d) ²⁰+16.6 (c 0.33, CHCl₃). ¹H NMR (400 MHz,CDCI₃): δ 0.69 (3H, s, H-18), 0.91 (3H, s, H-19), 3.37 (3H, s, H-2′,MOM), 3.53 (1H, tt, J₁=11.2, J₂=4.7, H-3), 4.45 (1H, tdd, J₁=7.6,J₂=6.0, J₃=1.6, H-16), 4.69 (2H, s, H-1′, MOM). ¹³C NMR (101 MHz,CDCl₃): δ 94.72 (C-3), 77.03 (C-16), 72.07, 55.30, 52.31, 52.29, 42.28,42.11, 40.80, 39.06, 37.44, 35.86, 35.54, 35.05, 33.74, 27.88, 27.33,26.85, 23.56, 20.59, 18.81. IR spectrum (CHCl₃): 3612 (OH); 2941 (CH₂);1040 (COCOC). MS (ESI) m/z: 359.3 (100%, M+Na). HR-MS (ESI) m/z: ForC₂₁H₃₅O₃ (M+Na) calcd: 359.2558; found: 359.2558. For C₂₁H₃₆O₃ (336.5)calcd: 74.95% C, 10.78% H; found: 74.68% C, 11.02% H.

Example 98(3R,5R,8S,9S,10S,13R,14S)-3-(Methoxymethoxy)-10,13-dimethylhexadecahydro-16H-cyclopenta[a]phenanthren-16-one(111)

Compound 110 (3.17 g, 9.4 mmol) was dissolved in freshly drieddichloromethane (150 ml). Then, pyridinium chlorochromate (16.7 g, 77.5mmol) in dried pyridine (10 ml) was added. The mixture was stirred underinert atmoshere at room temperature for 2 h. The reaction mixture wasfiltered through a small silica gel column (15 g), while washing withethyl acetate. The solvents were evaporated; the residue redissolved inethyl acetate and washed with aqueous citric acid (5%, 2×40 ml),saturated aqueous sodium chloride solution (50 ml), saturated aqueoussodium bicarbonate solution (2×30 ml), again saturated aqueous sodiumchloride solution (50 ml) and dried over anhydrous sodium sulfate.Evaporation of solvents under reduced pressure afforded 2.59 g (95%) of111: mp 107.9-108° C. (acetone/n-heptane), [α]_(D) ²⁰−134.7 (c 0.35,CHCl₃). ¹H NMR (400 MHz, CDCl₃): δ 0.86 (3H, s, H-18), 0.95 (3H, s,H-19), 3.37 (3H, s, H-2′, MOM), 3.54 (1H, ddd, J₁=15.8, J₂=11.1, J₃=4.7,H-3), 4.69 (2H, s, H-1′, MOM). ¹³C NMR (101 MHz, CDCl₃): δ 218.91 (C═O),94.78 (C-3), 56.12, 55.34, 51.91, 42.05, 40.68, 39.49, 39.40, 38.56,35.47, 35.25, 35.10, 33.71, 27.87, 27.13, 26.89, 23.49, 20.50, 18.23. IRspectrum (CHCl₃): 2937 (—CH₂—); 1737 (C═O); 1045, 1037 (COCOC). MS (ESI)m/z: 335.3 (32%, M+H) 273.2 (100%). HR-MS (ESI) m/z: For C₂₁H₃₅O₃ (M+H)calcd: 335.2586; found: 335.2579. For C₂₁H₃₄O₃ (334.5) calcd: 75.41% C,10.25% H; found: 75.66% C, 10.33% H.

Example 99(3R,5R,8S,9S,10S,13R,14S)-3-Hydroxy-10,13-dimethylhexadecahydro-16H-cyclopenta-[a]phenanthren-16-one(112)

To a solution of the protected derivative 111 (3.4 g, 10.2 mmol) inmethanol (95 ml) was added hydrochloric acid (1.8 ml, 37%) in methanol(30 m1). The reaction mixture was stirred under argon at roomtemperature overnight. Water was added (100 ml) and the mixtureconcentrated under reduced pressure. The product was extracted intoethyl acetate (3×70 ml), the combined organic phases washed withsaturated aqueous sodium bicarbonate, saturated aqueous sodium chloridesolution, dried over anhydrous sodium sulfate and the solventsevaporated under reduced pressure. Hydroxyketone 112 was obtained (3.17g, 93%): mp 135.8-136.7° C. (acetone/n-heptane), [α]_(D) ²⁰−167.4 (c0.33, CHCl₃). ¹H NMR (400 MHz, CDCl₃): δ 0.86 (3H, s, H-18), 0.96 (3H,s, H-19), 3.65 (1H, tt, J₁=10.8, J₂=4.7, H-3). ¹³C NMR (101 MHz, CDCI₃):δ 218.88 (C═O), 71.81, 56.12, 51.91, 42.01, 40.74, 39.48, 39.40, 38.56,36.55, 35.49, 35.22, 34.96, 30.64, 27.09, 26.93, 23.45, 20.51, 18.23. IRspectrum (CHCl₃): 2936 (CH₂); 1736 (C═O); 3609, 1032 (OH). MS (ESI) m/z:290.2 (100%, M). HR-MS (ESI) m/z: For C₁₉H₃₀O₂ (M+Na) calcd: 313.2138;found: 313.2137. For C₁₉H₃₀O₂(290.4) calcd: 78.57% C, 10.41% H; found:78.27% C, 10.36% H.

Example 100(3R,5R,8S,9S,10S,13R,14S)-10,13-Dimethyl-16-methylenhexadecahydro-1H-cyklo-penta[a]phenanthren-3-ol(113)

Compound 113 was prepared from compound 112 (800 mg, 2.76 mmol)analogously to the preparation of compound 66. Chromatography on, silicagel (1-5% acetone in petroleum ether) gave 756 mg (95%) of compound 113:mp 165.3-165.6° C. (acetone/n-heptane), [α]_(D) ²⁰−69.8 (c 0.35, CHCl₃).¹H-NMR (400 MHz, CDCl₃): δ 0.86 (3H, s, H-18), 0.96 (3H, s, H-19), 4.88(ddh, J₁=4.4, J₂=3.0, J₂=1.6, 1H), 3.63 (tt, J₁=11.1, J₂=4.7, 1H), 2.33(ddt, J₁=16.1, J₂=7.5, J₂=1.6, 1H). ¹³C NMR (101 MHz, CDCl₃): δ 151.39(═CH₂), 107.09 (C-16), 71.99 (C-3), 54.39, 49.61, 42.23, 40.89, 40.78,38.79, 36.63, 35.96, 35.50, 34.91, 33.54, 30.71, 27.29, 26.85, 23.53,20.92, 17.76. IR spectrum (CHCl₃): 3609 (OH); 3015, 1658 (═CI-1₂); 2934(CH₂). MS (ESI) m/z: 311.3 (100%, M+Na). HR-MS (ESI) m/z: For C₂₅H₃₈O₄Na(M+Na) called: 311.2345; found: 311.2344. For C₂₀H₃₂O (288.5) calcd:83.27% C, 11.18% H; found: 83.02% C, 11.07% H.

Example 101 Pyridinium(3R,5R,8S,9S,10S,13R,14S)-10,13-dimethyl-16-methylenhexadekhydro-1H-cyclopenta[a]phenanthren-3-yl3-sulfate (114)

Compound 114 was prepared according to General Procedure I—Preparationof C-3 Sulfate from compound 113 (83 mg, 0.29 mmol) affording sulfate114 (66 mg, 56%): mp 180-182° C. (chloroform), [α]_(D) ²⁰−41.9 (c 0.35,CHCl₃). ¹H NMR (400 MHz, CDCl₃): δ 0.70 (3H, s, H-18), 0.91 (3H, s,H-19), 2.30 (1H, dd, J₁=16.0, J₂=7.7), 4.45 (1H, tt, J₁=11.2, J₂=4.9H-3), 4.87 (2H, m, ═CH₂), 8.02 (2H, m, H-2′ and H-4′, pyridinium), 8.48(1H, t, J=8.6, H-3′, pyridinium), 8.99 (2H, d, J=5.2, H-1′ and H-5′,pyridinium). ¹³C NMR (101 MHz, CDCl₃): δ 218.89 (C-16), 178.74, 145.89(C-1′ and C-5′, pyridinium), 142.39 (C-3′, pyridinium), 127.31 (C-2′ andC-4′, pyridinium), 79.51 (C-3), 56.11, 51.94, 42.05, 40.68, 39.48,39.38, 38.57, 35.42, 35.21, 34.85, 33.42, 27.85, 26.85, 26.82, 23.68,20.40, 18.02. IR spectrum (CHCl₃):1736 (C═O); 1656 (C═C); 1460 (═CH,pyridine); 1263, 1171, 969, 947 (OSO₃). MS (ESI) m/z: 467.2 (100%, M+H-pyridine). HR-MS (ESI) m/z: For C₂₀H₃₁O₄S calcd: 367.1946; found:367.1945.

Example 1024-(((3R,5R,8S,9S,10S,13R,14S)-10,13-Dimethyl-16-methylenhexadecahydro-1H-cyclopenta[a]phenanthren-3-ypoxy)-4-oxobutarioicacid (115)

Compound 115 was prepared according to General Procedure II—Preparationof C-3 Hemisuccinate from compound 113 (95 mg, 0.33 mmol).Chromatography on silica gel (10-20% acetone in petroleum ether) gavecompound 115 (120.4 mg, 84.3%): mp 165-165.7° C. (acetone/n-heptane),[α]_(D) ²⁰−36.8 (c 0.29, CHCl₃). ¹H NMR (400 MHz, CDCl₃): δ 0.73 (3H, s,H-18), 0.94 (3H, s, H-19), 2.38-2.27 (2H, m, H-16a, H-16b), 2.72-2.55(4H, m, succinic acid), 4.76 (1H, tt, J₁=11.4, J₂=4.7, H-3), 4.89 (2H,ddq, J₁=4.2, J₂=2.9, J₃=1.5, ═CH₂), 4.58-4.65 (2H, m,), 4.74 (1H, tt,J₁=11.3, J₂=4.7). ¹³C NMR (101 MHz, CDCl₃): δ 177.07 (COOH), 171.82(COO), 151.29 (═CH₂), 107.14 (C-16), 75.11 (C-3), 54.37, 49.61, 42.04,40.89, 40.77, 38.75, 35.90, 35.16, 34.93, 33.51, 32.34, 29.43, 29.00,27.11, 26.72, 23.49, 20.94, 17.75. IR spectrum (CHCl₃): 3518 (OH, COOH,monomer); 3070, 1657 (═CH₂); 2674 (OH, COOH, dimer); 1752 (C═O, COOH,monomer); 1717 (C═O, COOH, dimer); 1727 (C═O, ester). MS (ESI) m/z:387.3 (100%, M−1). HR-MS (ESI) m/z: For C₂₄H₃₅O₄ (M−H) calcd: 387.2541;found: 387.2527. For C₂₄H₃₅O₄ (388.5) calcd: 74.19% C, 9.34% H; found:74.19% C, 9.34% H.

Preparation of Compounds 116-128 is Summarized in Table 1. Example 103(R)-5-benzyl 1-((3R ,5R ,8S,9S,10S,13S,14S)-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-3-yl)2-((tert-butoxycarbonyl)amino)pentanedioate (129)

Compound 129 was prepared from compound 48 (309 mg, 1.11 mmol)analogously to the preparation of compound 105. Chromatography on silicagel (3% acetone in petroleum ether) gave 214 mg (36%) of slightly impuredesired product and 361 mg (54%) of pure oily compound 129: [α]_(D)²⁰+17.8 (c 0.27, CHCl₃). ¹H NMR (400 MHz, CDCl₃): δ 0.68 (3H, s, H-18),0.93 (3H, s, H-19), 1.43 (9H, s, O¹Bu), 2.20 (1H, m, H-3a′), 2.44 (2H,m, H-4′), 4.28 (1H, m, H-2′), 4.84 (1H, m, H-3), 4.76 (1H, m, H-3), 5.11(3H, m, OCH₂Ph a NH). ¹³C NMR (101 MHz, CDCl₃): δ 172.59 (C-1′), 171.63(C-5′), 155.34 (NHCO), 135.80 (C-1, benzyl), 128.55 (2 ×C-3, benzyl),128.24 (C-4, benzyl), 128.18 (2 ×C-2, benzyl), 79.88 (O¹Bu), 75.85(C-3), 66.45 (OCH₂Ph), 54.50, 53.05, 41.95, 40.93, 40.73, 40.48, 38.98,36.18, 35.06, 34.73, 32.15, 30.32, 28.32, 28.0, 27.0, 26.70, 26.55,25.51, 23.32 (3 ×C, O¹Bu), 20.86, 20.59, 17.50. I{hacek over (C)}(CHCl₃): 3092, 3068 (CH, benzyl); 1730 (C═O, ester); 1713 (C═O, amide);1499 (NH, amide); 1260, 1235 (OCN, ester); 1165 (O¹Bu). MS (ESI) m/z:618.2 (100%, M+Na), 619.2 (40%, M+Na+1). HR-MS (ESI) m/z: ForC₃₆H₅₃NO₆Na [M+Na] calcd: 618.3765; found: 618.3763. For C₃₆H₅₃NO₆(595.2) calcd: 72.57% C, 8.97% H, 2.35% N; found: 72.52% C, 9.12% H,2.11% N.

Example 104(4S)-4-Amino-5-(((3R,5R,8S,9S,10S,13S,14S)-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-3-yl)oxy)-5-oxopentanoicacid (130)

Compound 130 was prepared from compound 129 (361 mg, 0.61 mmol)analogously to the preparation of compound 106 affording 186 mg (80%) of130: mp l 67-168° C., [α]_(D) ²⁰+24.4 (c 0.16, CHCl₃). ¹H NMR (400 MHz,CDCl₃+3 drops of MeOD): δ 0.61 (3H, s, H-18), 0.88 (3H, s, H-19), 2.40(2H, t, J=6.3, H-3′), 3.69 (1H, dd, J₁=8.1, J₂=3.4, H-2′), 4.75 (1H, m,H-3). ¹³C NMR (101 MHz, CDCI₃): δ 177.51 (COOH), 171.32 (COO), 76.61(C-3), 54.40, 53.24, 41.83, 40.79, 40.65, 40.31, 38.83, 36.04, 34.85,34.59, 33.10, 31.94, 27.37, 26.87, 26.57, 2638, 25.36, 23.14, 20.72,20.42, 17.32. IR spectrum (CHCl₃): 2646, 2179, 1609 (NH₃ ^(+);) 1748(C═O, ester); 1570 (COOH). MS (ESI) m/z: 406.3 (100%, M+1), 428.3 (99%,M+Na). HR-MS (ESI) m/z: For C₂₄H⁴⁰NO₄ [M+1] calcd: 406.2951, found:406.2951.

Example 1051-((3R,5R,8S,9S,10S,13S,14S)-10,13-Dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-3-yl)-5-oxopyrrolidine-3-carboxylicacid (131), mixture of isomers

Diisopropylethylamine (336 μI, 1.93 mmol) was added to a solution ofamine 57 (200 mg, 0.64 mmol) in nitroethane (10 ml) at room temperature.Then, the reaction mixture was heated at 105° C. for 22 h followed byheating at 125° C. for additional 20 h. The crude reaction mixture wasconcentrated and directly purified by column chromatography on silicagel (4:1:0.1 petroleum ether/acetone/acetic acid) affording compound 131(45 mg, 18%) as a mixture of carboxylic acids (1:1 according NMR): mp85-86.6° C., [α]_(D) ²⁰+20.1 (c 0.15, CHCl₃). ¹H NMR (600 MHz, CDCl₃): δ0.69 (3H, s, H-18), 0.95 (3H, s, H-19), 0.99 (1H, m, H-14), 1.09-1.465(2H, m, H-7), 1.16-1.735 (2H, m, H-12), 1.16-1.43 (2H, m, H-17),1.16-1.65 (2H, m, H-15), 1.27-1.42 (2H, m, H-11), 1.24-1.87 (2H, m,H-6), 1.26-1.91 (2H, m, H-4), 1.34 (1H, m, H-8), 1.34 (1H, m, H-9), 1.50(1H, m, H-5), 1.61 (2H, m, H-16), 2.75 (2H, m, NC(═O)CH₂CH(COOH)CH₂),3.25 (1H, m, NC(═O)CH₂CH(COOH)CH₂), 3.66 (2H, m, NC(═O)CH₂CH(COOH)CH₂),4.01 (1H, br, H-3). ¹³C NMR (150.9 MHz, CDCl₃): δ 176.48 (COOH), 172.09(NC(═O)CH₂CH(COOH)CH₂), 54.58 (C-14), 51.71 (C-3), 45.17(NC(═O)CH₂CH(COOH)CH₂), 42.51 (C-5), 40.94 (C-13), 41.01 (C-9), 40.50(C-17), 39.04 (C-12), 36.17 (C-8), 36.05 (NC(═O)CH₂CH(COOH)CH₂), 35.95(C-1), 34.73 (C-10), 34.56 (NC(═O)CH₂CH(COOH)CH₂), 30.15 (C-4), 27.09(C-6), 26.86 (C-7), 25.51 (C-15), 24.70 (C-2), 23.63 (C-19), 20.84(C-11), 20.58 (C-16), 17.52 (C-18). IR spectrum (CHCl₃): 3513 (OH), 2935(CH₂). 1754 (COOH), 1714 (C═O), 1674 (amide). MS (ESI) m/z: 1184.8 (40%,3M+Na), 797.5 (65%, 2M+Na), 410.3 (100%, M+Na), 388.3 (15%, M+H). HR-MS(ESI) m/z: for C₂₄H₃₇O₃NNa (M+Na) calcd: 410.2666, found 410.2671; forC₂₄H₃₈0₃N [M+H] calcd. 388.2846, found 388.2852.

Example 106(3S,5R,8R,9S,10S,13S,14S)-10,13-Dimethyl-17-methylenehexadecahydro-1H-cyclopenta[a]phenanthren-3-ol(132)

A solution of methyltriphenylphosphonium iodide (14.27 g, 35.20 mmol) indimethyl sulfoxide (80 ml) was stirred at room temperature for 20 minunder inert atmosphere. Then sodium hydride (50% in parafine oil, 1.44g, 35.97 mmol) was added. After 1 h of stirring, a solution of3beta-5beta-androstan-17-one (2.0 g, 6.89 mmol) in dimethyl sulfoxide(80 ml) was added and after 2.5 h of stirring at 70° C. under inertatmosphere, the reaction mixture was poured into brine. The precipitatedwhite solid was collected by filtration, washed with brine, dried andthe solvents were evaporated in vacuo. The residue was chromatographedon silica gel (5-10% ethyl acetate in petroleum ether) to afford 1.85 g(93%) of 132: mp 140.5-141.3° C. (acetone/n-heptane), [α]_(D) ²⁰+20.9 (c0.29, CHCl₃). ¹H NMR (400 MHz, CDCl₃): δ 0.77 (3H, s, H-18), 0.98 (3H,s, H-19), 4.11 (1H, m, H-3), 4.60-4.64 (2H, m, ═CH₂), ¹³C NMR (101 MHz,CDCl₃): δ 162.23 (CH═), 100.73 (═CH₂), 67.29 (C-3), 54.80, 44.40, 40.20,36.83, 36.12, 35.78, 35.46, 33.68, 30.17, 29.62, 28.01, 26.73, 2635,24.31, 24.07, 21.15, 18.69.1R spectrum (CHCl₃): 3616, 1028 (01-1); 1653(C═C). MS: ESI nez 311.3 (100%, M+Na). HR-MS (CI) m/z for C₂₀H₃₂O (M)calcd. 288.2453, found 288.2455. For C₂₀H₃₂O (288.5) calcd: 83.27% C,11.18% H; found: 83.22% C, 11.34% H.

Example 107(3S,5R,8S,9S,10S,13R,14S,17S)-10,13,17-Trimethylhexadecahydro-1H-cyclopenta[a]phenanthren-3-ol(133)

Compound 133 was prepared according to General Procedure V—CatalyticHydrogenation from compound 132 (718 mg, 2.49 mmol). Compound 133 (723mg, 99%): mp 145.6-146.2° C. (acetone/n-heptane), [α]_(D) ²+8.0 (c 0.27,CHCl₃). ¹H NMR(400 MHz, CDCl₃): δ 0.53 (3H, s, H-18), 0.82 (3H, d,J=6.8, H-20), 0.97 (3H, s, H-19), 4.11 (1H, m, H-3). ¹³C NMR (101 MHz,CDCl₃): δ 67.36, 56.13, 45.34, 42.36, 40.30, 37.94, 36.86, 36.03, 35.45,33.72, 30.40, 30.21, 28.01, 26.83, 26.64, 24.89, 24.11, 21.01, 13.99,12.20. IR spectrum (CHCl₃): 3616, 1030 (OH); 1379 (CH₃). MS: ESI m/z313.3 (100%, M+Na). HR-MS (ESI) m/z For C₂₀H₃₄ONa (M+Na) calcd.313.25019, found 313.25046. For C₂₀H₃₄O (290.5) calcd: 82.69% C, 11.80%H; found: 82.53% C, 11.41% H.

Example 108(3S,5R,8S,9S,10S,13R,14S,17S)-10,13,17-Trimethylhexadecahydro-1H-cyclopenta[a]phenanthren-3-yl4-methylbenzenesulfonate (134)

Compound 134 was prepared according to General Procedure VIII—Tosylationfrom compound 133 (0.84 g, 2.89 mmol). Compound 134 (1.12 g, 87%): mp112.6-113.8° C. (diethyl ether/n-heptane), [α]_(D) ²⁰+10.1 (c 0.30,CHCl₃). ¹H NMR (400 MHz, CDCl₃): δ 0.51 (3H, s, H-18), 0.81 (3H, d,J=6.8, H-20), 0.95 (3H, s, H-19), 2.44 (3H, s, CH₃-tosylate), 4.83 (1H,m, H-3), 7.32 (2H, d, J=8.2, tosylate), 7.78 (2H, d, J=8.2, tosylate).¹³C NMR (101 MHz, CDCl₃): δ 144.39 (C-1′, tosylate), 134.92 (C-4′,tosylate), 129.83 (C-1′, C-5′, tosylate), 127.75 (C-2′, C-6′, tosylate),81.08 (C-3), 56.05, 45.30, 42.32, 40.61, 37.85, 36.98, 35.95, 34.95,31.49, 30.35, 30.24, 26.43, 26.37, 25.95, 24.83, 23.79, 21.77, 20.95,13.96, 12.17. IR spectrum (CHCl₃): 1175 (SO₂); 903 (C-OTs). MS: ESI m/z467.3 (60%, M+Na), 911.7 (100%, 2M+Na). HR-MS (ESI) m/z for C₂₇H₄₀O₃NaS(M+Na) calcd: 467.25904, found 467.25907. For C₂₇H₄₀O₃S (444.67) calcd:72.53% C, 9.07% H; found: 73.08% C, 9.33% H.

Example 109(3R,5R,8S,9S,10S,13R,14S,17S)-3-Azido-10,13,17-trimethylhexadecahydro-1H-cyclopenta[a]phenanthrene(135)

Compound 135 was prepared according to General Procedure IX—Substitutionof Tosylate Protecting Group with Alkali Azide from compound 134 (2.05g, 4.62 mmol). Compound 135 (1.32 g, 91%): mp 59-59.5° C.(chloroform/methanol), [α]_(D) ²⁰+33.5 (c 0.31, CHCl₃). ¹H NMR (400 MHz,CDCl₃): δ 0.52 (3H, s, H-18), 0.82 (3H, d, J=6.8, H-20), 0.94 (3H, s,H-19), 3.31 (1H, m, H-3). ¹³C NMR (101 MHz, CDCl₃): δ 61.45 (C-3),55.88, 45.26, 42.65, 42.32, 41.02, 37.78, 36.19, 35.84, 34.95, 32.65,30.38, 27.27, 26.90, 26.70, 24.88, 23.65, 20.73, 13.99, 12.18. IRspectrum (CHCl₃): 2942, 2869 (CH₃); 2094 (N₃). MS: CI m/z 316.3(15%, M+1), 273.3 (100%, M−N₃), 287.3 (80%, M−N₂). HR-MS (CI) m/z for C₂₀H₃₃(M−N₃) calcd. 273.2584, found 273.2582. For C₂₀H₃₃N₃ (315.3) calcd:76.14% C, 10.54% H, 13.32% N; found: 76.48% C, 10.72% H, 13.05% N.

Example 109(3R,5R,8S,9S,10S,13R,14S,17S)-10,13,17-Trimethylhexadecahydro-1H-cyclopenta[a]phenanthren-3-aminehydrochloride (136)

Azide 135 (693 mg, 2.20 mmol) was dissolved in methanol (25 ml) andEtOAc (12 ml) and 5% palladium on CaCO₃ (80 mg) was added to thereaction mixture. The mixture was hydrogenated for 18 h under a slightoverpressure of hydrogen. The catalyst was then filtered off, thesolvent was evaporated in vacuo and the residue was dissolved in min.amount of ethanol and poured into 5% aqueous HC1 (100 ml). The productwas extracted with chloroform (3×20 ml), combined organic extracts weredried, the solvent was evaporated in vacuo affording 432 mg (60%) ofcompound 136: mp 284-287° C. (chloroform/diethyl ether),[α]_(D) ²⁰+24.3(c 0.30, CHCl₃).¹H NMR (400 MHz, CDCl₃): δ 0.51 (3H, s, H-18), 0.82 (3H,d, J=6.8, H-20), 0.95 (3H, s, H-19), 3.04 (1H, m, H-3). ¹³C NMR (101MHz, CDCl₃) δ 55.45, 52.12, 45.09, 42.52, 42.27, 40.86, 37.55, 36.24,35.42, 34.88, 31.86, 30.37, 27.00, 26.65, 26.37, 24.89, 23.61, 20.78,13.97, 12.15. IR spectrum (CHCl₃): 2978 (CH₃), 2940 (N⁺H₃and CH₂). MS:ESI m/z 290.3 (100%, M−C1. HR-MS (ESI) m/z for C₂₀H₃₆N (M−C1) calcd:290.28423, found 290.28425. For C₂₀H₃₆NCl (325.3) calcd: 73.69% C,11.13% H, 4.30% N; found: 72.42% C, 11.13% H, 4.03% N.

Example 110 Ethyl2-oxo-2-(((3R,5R,8S,9S,10S,13R,14S,17S)-10,13,17-trimethylhexadecahydro-1H-cyclopenta[a]phenanthren-3-yl)amino)acetate (137)

Compound 137 was prepared according to General Procedure XI—Reaction ofC-3 Amino Group with Ethyl Chlorooxoacetate from compound 136 (210 mg,0.64 mmol). Chromatography on silica gel (5% ethyl acetate in petroleumether) afforded 93 mg (37%) of oily compound 137: [α]_(D) ²⁰+41.7 (c0.31, CHCl₃). ¹H NMR (400 MHz, CDCl₃): δ 0.53 (3H, s, H-18), 0.82 (3H,d, J=6.8, H-20), 0.95 (3H, s, H-19), 1.38 (3H, t, J=7.1, CH₃-ethyl),3.75-3.87 (1H, m, H-3), 4.34 (2H, q, J=7.1, CH₂-ethyl), 6.96 (1H, d,J=8.5, N—H). ¹³C NMR (101 MHz, CDCl₃) δ 161.22, 155.77, 63.30, 56.06,50.22, 45.33, 42.51, 42.34, 41.12, 37.87, 36.18, 35.93, 34.85, 33.27,30.38, 27.56, 27.12, 26.73, 24.88, 23.71, 20.73, 14.16, 13.99, 12.18. IRspectrum (CHCl₃): 2868 (CH₃); 1696 (C═O). MS: ESI m/z 412.4 (55%, M+Na),801.9 (100%, 2M+Na). HR-MS (ESI) m/z for C₂₄H₃₉O₃NNa (M+Na) calcd:412.28222, found 48.28233. For C₂₄H₃₉O₃N (389.3) calcd: 73.99% C, 10.09%H, 3.60% N; found: 74.41% C, 10.13%H, 3.21%N.

Example 111 Sodium2-oxo-2(((3R,5R,8S,9S,10S,13R,14S,17S)-10,13,17-trimethylhexadecahydro-1H-cyclopenta[a]phenanthren-3-yl)amino)acetate(138)

A solution of NaOH (130 mg, 3.25 mmol) in MeOH (2 ml) was added dropwiseat 0° C. to a stirred solution of protected amide 137 (93 mg, 0.24 mmol)in MeOH (3 ml). Stirring was continued at 10° C. for 2 h and then thereaction mixture was poured into water, the precipitated white solid wascollected by filtration, washed with water and dried to afford 72 mg(79%) of amide 138: mp 330-334.6° C. (water), [α]_(D) ²⁰ insoluble inchloroform. ¹H NMR (400 MHz, DMSO-d₆): δ 0.50 (3H, s, H-18), 0.80 (3H,d, J=6.8, H-20), 0.90 (3H, s, H-19), signal of C-3 is overlapped by thesignal of DMSO. ¹³C NMR (101 MHz, DMSO-d₆): δ 165.10, 163.23, 55.25,48.57, 44.70, 42.19, 41.85, 37.23, 36.02, 35.66, 34.42, 32.78, 29.90,27.00, 26.84, 26.26, 24.46, 23.37, 20.22, 13.93, 11.95.1IR spectrum(KBr): 1668 (C═O); 1522 (amide). MS: ESI m/z 360.3 (100%, M−Na). HR-MS(ESI) m/z for C₂₂H₃₄O₃N (M−Na) calcd: 360.25442, found 360.25392. ForC₂₂H₃₄O₃NNa (383.2) calcd: 68.90% C, 8.94% H, 3.65% N; found: 67.85% C,9.17% H, 3.24% N.

Example 112 Methyl3-oxo-3-(3R,5R,8S,9S,10S,13R,14S,17S)-10,13,17-Trimethylhexadecahydro-1H-cyclopenta[a]phenanthren-3-yl)amino)propanoate(139)

Compound 139 was prepared according to General Procedure X—Reaction ofC-3 Amino Group with Methyl 3-Chloro-oxopropionate from compound 136(202 mg, 0.52 mmol). Chromatography on silica gel (8% acetone inpetroleum ether) afforded 258 mg (95%) of oily compound 139: mp >240° C.(acetone/n-heptane), [α]_(D) ²⁰+33.7 (c 0.35, CHCl₃). ¹H NMR (400 MHz,CHCl₃): δ 0.52(3H, s, H-18), 0.81 (3H, d, J=6.8, H-20), 0.94 (3H, s,H-19), 3.30 (2H, s, COCH₂CO), 3.75 (3H, s, OCH₃), 3.80 (1H, m, H-3). ¹³CNMR (101 MHz, CDCl₃): δ 170.33 (COOMe), 163.72 (NHCO), 55.87, 52.36,49.58, 45.17, 42.47, 42.18, 40.96, 40.92, 37.70, 36.04, 35.93, 34.72,33.48, 30.24, 27.73, 27.01, 26.58, 24.72, 23.57, 20.57, 13.83, 12.00.1IRspectrum (CHCl₃): 2939 (CH₂); 1723 (C═O); 1538, 1282 (amide and ester);1344 (CH₃). MS: ESI m/z 388.3 (100%, M−1). BR-MS (ESI) m/z for C₂₄H₃₈O₃N(M−H) calcd: 388.28572, found 388.28503. For C₂₄H₃₉O₃N (389.3) calcd:73.99% C, 10.09% H, 3.60% N; found: 73.58% C, 10.13% H, 3.28% N.

Example 1133-Oxo-3-(((3R,5R,8S,9S,10S,13R,14S,17S)-10,13,17-triinethylhexadecahydro-1H-cyclopenta[a]phenanthren-3-yl)amino)propanoicacid (140)

A solution of NaOH (36 mg, 0.90 mmol) in H₂O (1.5 ml) was added at 0° C.to a stirred solution of amide 139 (250 mg, 0.64 mmol) in EtOH (15 ml).Stirring was continued at room temperature for 2 h and then the reactionmixture was poured into water, acidified with 5% aqueous HCl to pH-2 andprecipitated amide 140 was filtered off, washed with water and dried.Compound 140 (32 mg, 92%): mp 147.8-149.4° C. (acetone/n-heptane),[α]_(D) ²⁰+41.4 (c 0.30, CHCl₃:MeOH, 1.795:0.043). ¹H NMR (400 MHz,CHCl₃): δ 0.51(3H, s, H-18), 0.81 (3H, d, J=6.8, H-20), 0.93 (3H, s,H-19), 3.23 (2H, s, COCH₂CO), 3.77 (1H, m, H-3). ¹³C NMR (101 MHz,CDCl₃): δ 170.32 (COOH, CONH), 56.04, 50.07, 50.00, 45.30, 42.54, 42.29,41.08, 37.85, 36.14, 35.96, 34.82, 33.31, 30.33, 27.56, 27.11, 26.71,24.83, 23.67, 20.68, 13.93, 12.12. IR spectrum (KBr): 3500, 3436(OH+NH); 2936 (CH₂); 1736, 1727 (COOH). MS: ESI m/z 374.2 (85%, M−1),(100%, M - COOH). HR-MS (ESI) m/z for C₂₃H₃₆O₃N (M−H) calcd: 374.27007,found 374.26954. For C₂₃H₃₇O₃N (375.2) calcd: 73.56% C, 9.93% H, 3.73%N; found: 73.41% C, 10.17% H, 3.23% N.

Example 114(3S,5R,8R,9S,10S,13R,14S)-17-((R)-Sec-butyl)-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-3-ol(143)

L-Selectride (1M in THF, 2.16 ml) was added dropwise under inertatmosphere to a cooled solution (−78° C.) ofR-sec-butyl-5beta-androstan-3-one 142 (600 mg, 1.8 mmol) in anhydrousTHF (50 ml). After 1 hr stirring at −78° C., water (5 ml) was added andthe mixture was allowed to attain room temperature. Then, an aqueoussolution of sodium hydroxide (6M, 5 ml) and an aqueous H₂O₂ solution (5ml, 30%) were added, and the reaction mixture was stirred for 30 min.The mixture was poured into cold water, the product was extracted withEtOAc (2×50 ml) and water phase was extracted again with ethyl acetate(30 ml). Combined extracts were washed with an aqueous solution ofhydrochloric acid (5%), saturated solution of sodium hydrogen carbonate,and brine. Solvent was dried over anhydrous sodium sulfate andevaporated. Column chromatography (10% EtOAc in petroleum ether) gavecompound 143 (520 mg, 87%): mp 151-153° C. (acetone/n-heptane), [α]_(D)²⁰+17 (c 0.20, CHCl₃). ¹H NMR (400 MHz, CDCl₃): δ 0.65 (3H, s, H-18),0.81 (3H, t, J=7.4, H-23), 0.89 (3H, d, J=6.6, H-21),0.97 (3H, s, H-19),4.10 (1H, m, H-3). ¹³C NMR (101 MHz, CDCl₃): δ 67.36 (C-3), 56.82,56.01, 42.84, 40.42, 39.92, 37.15, 36.76, 35.82, 35.33, 33.63, 30.03,28.44, 28.34, 27.96, 26.83, 26.46, 24.43, 24.08, 21.27, 18.21, 12.23,10.48. IR spectrum (CHCl₃): 3616, 1029 (OH); 1381 (CH₃). MS: ESI m/z332.3 (100%, M), 3153 (78%, M−17), 313.3 (71%, M−19), 299.3 (24%, M−33).HR-MS (ESI) m/z for C₂₃H₄₀O (M) calcd: 332.3079, found 332.3082.

Example 115(3S,5R,8R,9S,10S,13R,14S)-17-((R)-Sec-butyl)-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-3-yl4-methylbenzenesulfonate (144)

Compound 144 was prepared according to General Procedure VIII—Tosylationfrom compound 143 (827 mg, 2.5 mmol). Compound 144 (1.1 g, 87%): mp103-105° C. (benzene, decomposition), [α]_(D) ²⁰+19.3 (c 0.32, CHCl₃).¹H NMR (400 MHz, CDCl₃): δ 0.62 (3H, s, H-18), 0.80 (3H, t, J=7.4,H-23), 0.87 (3H, d, J=6.5, H-21), 0.93 (3H, s, H-19), 2.44 (3H, s,CH₃-tosylate), 4.82 (1H, m, H-3), 7.32 (2H, m, tosylate), 7.78 (2H, d,J=8.3, tosylate). ¹³C NMR (101 MHz, CDCl₃):δ 144.40 (C-1′, tosylate),134.81 (C-4′, tosylate), 129.83 (C-1′, C-5′, tosylate), 127.74 (C-2′,C-6′, tosylate), 81.10 (C-3), 56.69, 55.91, 42.79, 40.47, 40.19, 37.09,36.84, 35.68, 34.77, 31.41, 30,08, 28.39, 28.28, 26.34, 26.22, 25.91,24.32, 23.75, 21.79, 21.18, 18.17, 12.18, 10.46. IR spectrum (CHCl₃):2940 (CH₃); 1175 (SO₂); 905 (C—OTs). ESI m/z 314.3 (100%, M−p-TsOH).HR-MS (ESI) m/z for C₃₀H₄₆O₃NaS (M+Na) calcd: 509.2590, found 509.2591.

Example 116(3R,5R,8R,9S,10S,13R,14S)-3-Azido-17-((R)-sec-butyl)-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthrene(145)

Compound 145 was prepared according to General Procedure IX—Substitutionof Tosylate Protecting Group with Alkali Azide from compound 144 (1.1 g,2.3 mmol). Compound 145 (770 mg, 95%): mp 99-101° C. (benzene), [α]_(D)²⁰+45.2 (c 0.10, CHCl₃). ¹H NMR(400 MHz, CDCl₃): δ 0.64 (3H, s, H-18),0.82 (3H, t, J=7.4, H-23), 0.89 (3H, d, J=6.5, H-21), 0.93 (3H, s,H-19), 3.31 (1H, tt, J=11.8, J=4.5, H-3), ¹³C NMR (101 MHz, CDCl₃): δ61.44, 56.56, 55.91, 42.78, 42.55, 40.66, 40.24, 37.15, 35.96, 35.71,34.80, 32.61, 28.42, 28.31, 27.26, 26.89, 26.51, 24.37, 23.61, 20.99,18.19, 12.19, 10.48. IR spectrum (CHCl₃): 2942, 2868 (CH₃) 2094 (N₃).MS: CI m/z 330.3(100%, M−N₂). FIR-MS (CI) m/z for C₂₃H₄₀N (M−N₂) calcd:330.3155, found 330.3156.

Example 117(3R,5R,8R,9S,10S,13R,14S)-174((R)-Sec-butyl)-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-3-aminehydrochloride (146)

Compound 146 was prepared from compound 145 (770 mg, 2.2 mmol)analogously to the preparation of compound 136 affording 790 mg (98%) of146: mp 301-302° C. (ethanol, decomposition), [α]_(D) ²⁰+30.8c (c 0.10,CHCl₃). ¹H NMR (400 MHz, CDCl₃): δ 0.61 (3H, s, H-18), 0.78 (3H, t,J=7.4, H-23), 0.86 (3H, d, J=6.5, H-21), 0.91 (3H, s, H-19), 3.00 (1H,ddt, J=11.8, J=8.5, J=4.3, H-3). ¹³C NMR (101 MHz, CDCl₃): δ 56.48,55.84, 51.50, 42.70, 42.25, 40.46, 40.16, 37.10, 35.89, 35.37, 34.62,32.47, 28.35, 28.24, 26.98, 26.83, 26.42, 24.32, 23.47, 20.93, 18.10,12.10, 10.41. IR spectrum (CHCl₃): 3437 (NH₂); 3192, 3011, 2786 (NH₃ ⁺).MS: ESI m/z 330.3 (100%, M−HCl—H). HR-MS (ESI) m/z for C₂₃H₄₂N (M−CI)calcd: 332.3317, found 332.3318. For C₂₃H₄₂NCl (368.0) calcd: 75.06% C,11.50% H, 3.81% N; found: 74.77% C, 11.56% H, 3.67% N.

Example 118 Ethyl2-(((3R,5R,8R,9S,10S,13R,14S)-17-((R)-sec-butyI)-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-3-yl)amino)-2-oxoacetate(147)

Compound 147 was prepared according to General Procedure XI—Reaction ofC-3 Amino Group with Ethyl Chlorooxoacetate from compound 146 (178 mg,0.48 mmol). Chromatography on silica gel (1-5% ethyl acetate inpetroleum ether) afforded 107 mg (51%) of oily compound 147: [α]_(D)²⁰+49.5 (c 0.34, CHCl₃). ¹H NMR (400 MHz, CDCI₃): δ 0.64 (3H, s, H-18),0.82 (3H, t, J=7.1, H-23), 0.89 (3H, d, J=6.5, H-21), 0.94 (3H, s,H-19), 1.38 (3H, t, J=7.1, CH₃- ethyl), 3.70-3.91 (1H, m, H-3), 4.34(2H, q, J=7.1, CH₂-ethyl), 6.96 (1H, d, J=8.3, N—H). ¹³C NMR (101 MHz,CDCl₃): δ 161.21 (COOMe), 155.77 (CONH), 63.30 (C-3), 56.74, 56.01,50.23, 42.81, 42.42, 40.74, 40.34, 37.15, 35.96, 35.80, 34.70, 33.25,28.43, 28.32, 27.57, 27.12, 26.54, 24.37, 23.68, 21.00, 18.20, 14.16,12.20, 10.49. IR (CHCl₃): 3539 (OH); 3405 (NH); 2935 (CH); 2867 (CH₃);1696 (C═O). MS: ESI m/z 432.4 (11%, M+1), 454.4 (100%, M+Na), 885.8(85%, 2M+Na). HR-MS (ESI) mz for C₂₇H₄₆O₃N (M+H) calcd: 432.34722, found432.34730.

Example 118 Sodium2-(((3R,5R,8R,9S,10S,13R,14S)-17-((R)-sec-butyl)-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-3-yl)amino)-2-oxoacetate(148)

Compound 148 was prepared from compound 147 (95 mg, 0.22 mmol)analogously to the preparation of compound 138 affording 42 mg (45%) of148: mp 182-184° C. (water, decomposition), [α]_(D) ²⁰+47.5 (c 0.14,DMSO). ¹H NMR (400 MHz, DMSO-d₆): δ 0.61 (3H, s, H-18), 0.79 (3H, t,J=7.3, H-23), 0.87 (3H, d, J=6.9, H-21), 0.88 (3H, s, H-19), signal forH-3 is overlapped by the peak of DMSO. ¹³C NMR (101 MHz, DMSO-d₆): δ167.01 (COONa), 16238 (CONH), 56.23, 55.49, 49.12, 42.29, 41.85, 39.85,36.52, 35.63, 35.27, 34.18, 31.97, 27.89, 27.80, 26.66, 26.27, 26.05,25.54, 23.89, 23.16, 20.47, 18.01, 11.94, 10.25. IR spectrum (KBr):3425, 3415 (NH); 1760 (C═O); 1640 (CO₂). MS: ESI m/z 402.3 (100%, M−Na).HR-MS (ESI) m/z for C₂₅H₄₀O₃N (M−Na) calcd: 402.30137, found 402.30106.

Example 1193-(((3R,5R,8R,9S,10S,13R,14S)-17-((R)-Sec-butyl)-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-3-yl)amino)-3-oxopropanoicacid (149)

Compound 149 was prepared according to General Procedure X—Reaction ofC-3 Amino Group with Methyl 3-Chloro-oxopropionate (affording themixture of amides in an inseparable mixture of keto and enol forms),followed by the deprotection reaction analogously to the procedure forcompound 140, from compound 146 (190 mg, 0.52 mmol). Compound 149 (65mg, 30% after 2 steps): mp 163-165° C. (acetone/n-heptane), [α]_(D)²⁰+53.3 (c 0.30, CHCl₃). ¹H NMR (400 MHz, CHCl₃): δ 0.64 (3H, s, H-18),0.82 (3H, t, J=7.4, H-23), 0.89 (3H, d, J=6.5, H-21), 0.94 (3H, s,H-19), 3.28 (2H, s, H-2′),3.83 (H, tdt, J=12.1, J=8.5, J=4.4, H-3), 6.13(d, H, J=7.8, NH). ¹³C NMR (101 MHz, CDCl₃): δ 168.32 (COO), 168.08(CONH), 56.76, 56.03, 50.61, 42.80, 42.45, 40.75, 40.34, 38.43, 37.14,35.93, 35.79, 34.70,33.33, 28.43, 28.32, 27.64, 27.10, 26.55, 24.3,23.65, 20.98, 18.20, 12.20, 10.50. IR spectrum (far): 3435,1631 (NH);1730, (C═O). MS: ESI m/z 833.6 (77%, 2M−1), 416.3 (100%, M−1), 372.3(11%, M-COOH). HR-MS (ESI) m/z for C₂₆H₄₂O₃N (M−H) calcd: 416.31702,found 416.31619.

Example 120(3S,5R,8R,9S,10S,13S,14S,Z)-17-ethylidene-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-3-oland(3S,5R,8R,9S,10S,13S,14S,E)-17-ethylidene-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-3-ol,mixture of E/Z isomers (150)

A solution of Ph₃PEtBr (6.386 g, 17.2 mmol) in dimethyl sulfoxide (30ml) was stirred at rt for 20 min under inert atmosphere and then NaH(50% in parafine oil, 688 mg, 17.2 mmol) was added. Stirring continuedfor 1 h, then a solution of 3beta-hydroxy-5beta-androstan-17-one (1.0 g,2.9 mmol) in dimethyl sulfoxide (18 ml) was added and after 15 h ofstirring at 60° C., aqueous solution of ammonium chloride was added. Theproduct was extracted with chloroform; combined organic extracts werewashed with brine, and dried. Solvents were evaporated and the residuewas purified by chromatography on silica gel (5-10% ethyl acetate inpetroleum ether) to afford 810 mg (78%) of compound 150 as mixture of Eand Z isomers (1.5:8.5). Z-isomer: mp 157-158° C. (acetone/n-heptane),[α]_(D) ²⁰+30.6 (c 0.32, CHCl₃). ¹H NMR (400 MHz, CDCl₃): δ 0.86 (3H, s,H-18), 0.97 (3H, s, H-19), 1.65 (3H, dt, J₁=7.2, J₂=2.0, H-21), 4.11(1H, m, H-3), 5.11 (1H, qt, J₁=7.1, J₂=2.0, H-20).¹³C NMR (101 MHz,CDCl₃): δ 150.64 (CH═CH—CH₃), 113.33 (CH═CH—CH₃), 67.31, 56.59, 44.63,39.98, 37.61, 36.73, 35.34, 33.68, 31.66, 30.06, 28.01, 26.76, 26.30,24.55, 24.02, 21.43, 17.04, 13.25. IR spectrum (CHCl₃): 3036 (═CH); 1673(C═C); 995 (C—OH). MS: CI m/z 302.3 (50%, M). HR-MS (CI) m/z for C₂₁H₃₄O(M) calcd: 302.2610, found 302.2608.

Example 1212-((3R,5R,8R,9S,10S,13S,14S,Z)-17-ethylidene-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-3-yl)isoindoline-1,3-dione(151)

A mixture of triphenylphosphine (409 mg, 1.56 mmol), phathalimide (230mg, 1.56 mmol) and compound 150 (360 mg, 1.2 mmol) in anhydrous THF (10ml) was stirred under inert atmosphere in ice bath for 1 h. Then,diisopropyl azodicarboxylate (DIAD, 0.31 ml, 1.56 mmol) was added andstirring continued for 18 h at rt. Distilled water was added to quenchthe reaction, THF was evaporated and the reaction mixture was extractedwith chloroform, washed with water, and dried. The solvent wasevaporated and the residue was purified by chromatography on silica gel(3-5% ethyl acetate in petroleum ether) to afford 320 mg (63%) ofphtalimide 151: mp 196-198° C. (methanol/dichlormethane)

[α]_(D) ²⁰+80.7 (c 0.24, CHCl₃). ¹H NMR (400 MHz, CDCl₃): δ 0.88 (3H, s,H-18), 0.97 (3H, s, H-19), 1,66(3H, dt, J₁=7.2, J₂=2.0, H-21), 4.19 (1H,m, H-3), 5.12 (1H, qt, J₁=7.2, J₂=2.0, H-20), 7.68-7.70 (2H, m),7.79-7.82 (2H, m). ¹³C NMR (101 MHz, CDCl₃): δ 168.55 (2×C), 150.43(CH═CH—CH₃), 133.78 (2×C), 132.12 (2×C), 122.95 (2×C), 113.16(CH═CH—CH₃), 56.09, 51.28, 44.47, 43.09, 40.56, 37.31, 36.62, 35,36,34.68, 31.50, 29.78, 27.07, 26.23, 24.41, 24.26, 23.40, 21.09, 16.95,13.14. IR (CHCl₃): 3030 (═CH); 1678 (C═C). MS: CI mz 431.3 (90%, M),432.3 (100%, M+1). HR-MS (CI) m/z for C₂₉H₃₈O₂N (M+H) calcd: 432.2903,found 432.2904.

Example 121(3R,5R,8R,9S,10S,13S,14S,Z)-17-Ethylidene-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-3-aminehydrochloride (152)

To a stirring solution of phtalimide 151 (115 mg, 0.26 mmol) in methanol(20 ml), hydrazine hydrate (85%, 3 ml) was added and reaction mixturewas refluxed for 2 h. Then, aqueous solution of NaOH (6 N, 20 ml) wasadded and stirred for 0.5 h, extracted with dichloromethane, washed withwater and dried. The residue was dissolved in min. amount of EtOH andpoured into 5% aqueous HCl (50 ml). The hydrochloride amine wasextracted with chloroform, combined organic extracts were dried andsolvent was evaporated under reduced pressure to obtain oily amine 152(85 mg, 94%): [α]_(D) ²+47.8 (c 0.33, MeOH). ¹H NMR (400 MHz, CDCl₃): δ0.89 (3H, s, H-18), 1.01 (3H, s, H-19), 1.65 (3H, dt, J₁=7.2, J₂=2.0,H-21), 3.11 (1H, m, H-3), 5.11 (1H, qt, J₁=7.2, J₂=2.1 H-20). ¹³C NMR(101 MHz, MeOD): δ 151.25 (CH═CH—CH₃), 114.45 (CH═CH—CH₃), 57.74, 52.32,45.58, 43.24, 41.86, 38.69, 36.63, 36.01, 35.64, 32.44, 32.37, 27.88,27.32, 26.73, 25.38, 23.68, 22.07, 17.25, 13.40. IR spectrum (CHCl₃):3436, 1617 (amine); 3013 (═CH); 1673 (C═C); 995 (C—OH). MS: ESI m/z302.3 (80%, M-Cl). HR-MS (ESI) m/z for C₂₁H₃₆N (M-C1) calcd: 302.28423,found 302.28433.

Example 121 Ethyl2-(((3R,5R,8R,9S,10S,13S,14S,Z)-17-ethylidene-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-3-yl)amino)-2-oxoacetate(153)

Compound 153 was prepared according to General Procedure XI—Reaction ofC-3 Amino Group with Ethyl Chlorooxoacetate from compound 152 (100 mg,034 mmol). Chromatography on silica gel (3-5% ethyl acetate in petroleumether) to afford of protected amide 153 (105 mg, 88%) as an inseparablemixture of keto and enol forms: ¹H NMR (400 MHz, CDCl₃): δ 0.86 (3H, s,H-18), 0.96 (3H, s, H-19), 1.38 (3H, t, J=7.1, H-ethyl), 1.65 (3H, dt,J₁=7.2, J₂=2.0, H-21), 3.81 (1H, m, H-3), 4.34 (2H, q, J=7.1, H-ethyl),5.12 (1H, qt, J₁=7.2, J₂=2.1, H-20), 6.96 (1H, d, J=6.9, H—NH).

Example 1222-(((3R,5R,8R,9S,10S,13S,14S,Z)-17-Ethylidene-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-3-yl)amino)-2-oxoaceticacid (154)

A solution of NaOH (110 mg, 2.75 mmol) in MeOH (2 ml) was added dropwiseat 0° C. to a stirred solution of protected amide 153 (110 mg, 0.27mmol) in Me0H (3 ml). Stirring was continued at 10° C. for 2 h and thenthe reaction mixture was poured into water, acidified with 5% aqueousHCl to pH-2 and extracted with ethyl acetate. The combined organicextracts were washed with brine, dried and the solvents were evaporatedin vacuo to afford 90 mg (88%) of amide (E/Z, 2:8) 154: ¹H NMR (400 MHz,CDCl₃): δ 0.86 (3H, s, H-18), 0.97 (3H, s, H-19), 1.65 (3H, dt, J₁=7 .1,J₂=2.0, H-21), 3.76 (1H, m, H-3), 5,12 (1H, qt, J₁=7.2, J₂=2.1, H-20),7.14 (1H, d, J=6.9, H—NH). ¹³C NMR (101 MHz, CDCl₃): δ 160.05 (COOH),156.65 (CONH), 150.35 (CH═CH—CH₃), 113.50 (CH═CH—CH₃), 56.44, 51.27,44.56, 42.43, 40.81, 37.48, 35.68, 35.48, 34.77, 33.06, 31.63, 27.40,27.03, 26.37, 24.55, 23.61, 21.16, 17.02, 13.26. IR spectrum (CHCl₃):3422, 1690 (amide); 3013 (═CH); 1669 (C═C); 1763 (C═O). MS: ESI m/z396.4 (100%, M+Na). HR-MS (ESI) m/z for C₂₃H₃₆NO₃ (M+H) calcd:374.26897, found 374.26910.

Example 1223-(((3R,5R,8R,9S,10S,13S,14S,Z)-17-Ethylidene-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-3-yl)amino)-3-oxopropanoicacid (155)

Compound 155 was prepared according to General Procedure X—Reaction ofC-3 Amino Group with Methyl 3-Chloro-oxopropionate from compound 152(150 mg, 0.44 mmol). Purification by column chromatography (3-5% ethylacetate in petroleum ether) afforded the mixture of amides in aninseparable mixture of keto and enol forms (130 mg, 73%). A solution ofNaOH (28 mg, 0.69 mmol) in H₂O (1.5 ml) was added at 0° C. to a stirredsolution of protected amides (130 mg, 0.34 mmol) in THF (1.5 ml).Stirring was continued at room temperature for 2 h and then the reactionmixture was poured into water, acidified with 5% aqueous HCl to pH-2 andextracted with ethyl acetate. The combined organic extracts were washedwith brine, dried and the solvents were evaporated in vacuo. The residuewas chromatographed on silica gel (30% acetone in petroleum ether with1% of TEA) to afford 69 mg (46%) of amide (E/Z, 2:8) 155: ¹H NMR (400MHz, CDCl₃): δ 0.85 (3H, s, H-18), 0.94 (3H, s, H-19), 1.64 (3H, dt,J₁=7.3, J₂=2.0, H-21), 3.23 (2H, s, COCH₂CO), 3.78 (1H, m, H-3),5.11(1H, qt, J₁=7.2, J₂=2.1, H-20), 7.14 (1H, d, J=6.9, H—NH). ¹³C NMR(101 MHz, CDCl₃): δ 167.91, 167.86, 150.48, 113.42, 56.47, 49.99, 45.32,44.55, 42.46, 40.72, 37.51, 35.90, 35.46, 34.78, 33.39, 31.64, 27.67,27.12, 26.42, 24.55, 23.65, 21.14, 16.99, 13.27. IR spectrum (CHCl₃):1743 (C═O); 1662 (C═C); 1646, 1636, 1540 (amide). MS: ESI m/z 386.4(20%, M−1), 342.4 (100%, M-COOH). HR-MS (ESI) m/z for C₂₄H₃₆NO₃ (M−H)calcd: 386.27007, found 386.26989.

The method used for the preparation of compound 8 was used in order toprepare additional compounds listed in Table 1:

TABLE 1 MS (m/z), Optical Molecular ion Melting Rotation withoutpyridinium Point [α]_(D) ¹H-NMR peaks: (m/z = 80.1), Compound (° C.)(20° C.) H-18; H-19; H-3 Relative Intensity Pyridinium 155-157 +41.60.73; 0.91; 4.44 311.3; 100% (3R,5R,8R,9S,10S,13S,14S)-10,13-dimethyl-17- methylenehexadecahydro-1H- cyclopenta[a]phenanthren-3-yl 3-sulfate (116) Pyridinium 174-175 −21.6 0.51; 0.91; 4.46 369.2; 100%(3R,5R,8S,9S,10S,13R,14S,17S)- 10,13,17-trimethylhexadecahydro-1H-cyclopenta[a]phenanthren-3-yl 3- sulfate (117) Pyridinium 152-155−0.5 N/A; 0.85; 4.46 355.2; 100% (3R,5R,8S,9S,10S,13S,14R,17R)- 0.75(17α-Me) 10,17-dimethylhexadecahydro-1H- cyclopenta[a]phenanthren-3-yl3- sulfate (118) Pyridinium 155-158 +26.9 N/A; 0.85; 4.45 355.2; 100%(3R,5R,8S,9S,10S,13R,14R,17S)- 0.90 (17β-Me)10,17-dimethylhexadecahydro-1H- cyclopenta[a]phenanthren-3-yl 3- sulfate(119) Pyridinium 182-184 +61.5 0.53; 0.86; 4.47 383.1; 100%(3R,5R,8S,9S,10S,13R,14S,17S)-17- ethyl-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-3-yl 3- sulfate (120) Pyridinium 169-172+42.0 0.52; 0.90; 4.45 395.2; 100% (3R,5R,8R,9S,10S,13S,14S,17R)-10,13-dimethyl-17-(prop-1-en-2- yl)hexadecahydro-1H-cyclopenta[a]phenanthren-3-yl 3- sulfate (121) Pyridinium 190-194 +26.20.62; 0.82; 4.46 397.2; 100% (3R,5R,8R,9S,10S,13R,14S,17R)-17-isopropyl-10,13- dimethylhexadecahydro-1H- cyclopenta[a]phenanthren-3-yl3- sulfate (122) Pyridinium 186-188 +13.8 0.62; 0.81; 4.46 411.4; 100%(3R,5R,8R,9S,10S,13R,14S,17R)-17- ((R)-sec-butyl)-10,13-dimethylhexadecahydro-1H- cyclopenta[a]phenanthren-3-yl 3- sulfate (123)Pyridinium 118-121 +30.5 3.37 a 3.63; 0.84; 397.1; 100%(3S,3aS,5bR,7aR,9R,11aS,11bS,13aR)- 4.47 3,11a-dimethylhexadecahydro-1H,3H-naphtho[2′,1′:4,5]indeno[1,7a- c]furan-9-yl 9-sulfate (124)Pyridinium 177-179 −13.8 0.89; 1.05; 4.45 383.3; 100%(2R,4aS,4bS,6aS,10bR,12aR)-4a,6a- dimethyl-7-oxooctadecahydrochrysen-2-yl 2- sulfate (125) Pyridinium 192-194 +19.00.66; N/A; 4.42 341.2; 100% (3R,5R,8R,9R,10S,13S,14S)-13-methylhexadecahydro-1H- cyclopenta[a]phenanthren-3-yl 3- sulfate (126)Pyridinium 167-169 +8.2 0.66; 0.77; 4.74 458.3; 50%(3R,5S,8R,9R,10S,13S,14S)-10,13- (M + Na − dimethylhexadecahydro-1H-pyridinium) cycopenta[a]phenanthren-3-yl 3- sulfate (127) Pyridinium(2S,4aR,4bR,8aS,10aS)-4a- 147-149 −24.1 N/A; 0.86; 4.47 301.0; 100%methyltetradecahydrophenanthren-2- yl 2-sulfate (128)

Biological Activity—Cell Cultures

Degree of inhibition of activated NMDA receptor by amphiphilic compoundswas measured in vitro electrophysiologically on cultivated HEK293 cells(Human Embryonic Kidney 293 cells) 24-48 h after the transfection withDNA plasmids, coding NR1-1a and NR2B subunit of NMDA receptor.Transfected cells were identified by means of fluorescent green protein(GFP) fluorescence. Its genus was transfected together with the bothreceptor subunit genes.

Steroid-containing solutions were prepared from fresh solution (20mmol.l⁻¹, of steroid dissolved in dimethyl sulfoxide, DMSO), which wasadded to the extracellular solution containing 1 mmol.l⁻¹ glutamic acidand 10 μmol.l⁻¹ of glycine. Identical concentrations of DMSO were addedto all other extracellular solutions.

Current responses produced by extracellular application of glutamic acidsolution (1 mmol.l⁻¹) were measured from the whole cell by patch-clamptechnique, which is used for the study of transport of charged particlesthrough model and also natural biological membranes. The currents weremeasured at membrane potential maintained at −60 mV and +60 mV. Steroidcompounds studied lowered response amplitude elicited by glutamic acid.Application of 10 μmol.l⁻¹ steroid solution the mean inhibition effectreached 65 -70%. It can be compared with 100 μmol.l⁻¹ of endogenousneurosteroid 5beta-pregnanolon-3alpha-yl 3-sulfate, which inhibitedresponses elicited by NMDA receptor to 67%.

Effect of Amphiphilic Compounds on Recombinant NMDA Receptors

HEK293 cells (American Type Culture Collection, ATTC No. CRL1573,Rockville, Md.) were cultivated in Opti-MEM® I media (Invitrogen) withaddition of 5% fetal bovine serum at 37° C. and transfected withNR1-1a/NR2B/GFP plasmids, as described in the scientific literature(Neuroscience 151, 428-438, 2008). Same amounts (0.3 μg) of cDNA codingNR1, NR2 and GFP (green fluorescent protein) (pQBI 25, Takara, Japan)were mixed with 0.9 μl of Matra-A Reagent (IBA, Göttingen, Germany) andadded to confluent HEK293 cells cultivated in v 24-pit cultivatingplate. After trypsination, the cells were re-suspended in Opti-MEM® Icontaining 1% fetal bovine serum. Subsequently, 20 mmol.l⁻¹ MgCl₂, 1mmol l⁻¹ D,L-2-amino-5-phosphonopentanoic acid, 3 mmol.l⁻¹ kynurenicacid was added to the mixture and cells were inoculated on thepolylysine-coated glass plates having 25 mm in diameter. The followinggenes coding NMDA receptor subunits were used for transfection: NR1-1 a(GenBank accession No. U08261) and NR2B (GenBank accession No. M91562).

HEK293 Cultured cells were used for electrophysiological investigationswith a latency of 16-40 h after transfection. Whole-cell currents weremeasured by patch-clamp amplifier (Axopatch 1D; Axon Instruments, Inc.Foster City, USA) after capacitance and serial resistance (<10 MΩ)compensation to 80-90%. Agonist-induced responses were filtered to 1 kHz(8-pole Bessel filter; Frequency Devices, Haverhill, USA), digitizedwith sampling frequency of 5 kHz and analyzed by pClamp version 9software (Axon Instruments, USA). Micropipettes made of borosilicateglass were filled with intracellular solution, containing 125 mmol.l⁻¹D-gluconic acid, 15 mmol.l⁻¹ cesium chloride, 5 mmol.l⁻¹ EGTA, 10mmol.l⁻¹ HEPES buffer, 3 mmol.l⁻¹ magnesium chloride, 0.5 mmol.l⁻¹calcium chloride and 2 mmol.l⁻¹ magnesium-salt of ATP (pH adjusted to7.2 by cesium hydroxide solution). Extracellular solution (ECS)contained 160 mmol.l⁻¹ sodium chloride, 2.5 mmol.l⁻¹ potassium chloride,10 mmol.l⁻¹ HEPES, 10 mmol.l⁻¹ glucose, 0.2 mmol.l⁻¹ EDTA a 0.7 mmol.l⁻¹calcium chloride (pH adjusted to 7.3 by sodium hydroxide solution).Glycine was added to both testing and control solution. Moreover,bicuculline (10 μmol.l⁻¹) and tetrodotoxin (0.5 μmol.l⁻¹) was added tohippocampal cultures. Steroid-containing solutions were prepared fromfresh solution (20 mmol.l⁻¹) of steroid dissolved in dimethyl sulfoxide(DMSO). Same concentrations of DMSO were used in all extracellularsolutions. Control and experimental solutions were applied viamicroprocessor-controlled perfusion system with approx. rate of solutionexchange in areas adjacent to cells reaching ˜10 ms.

Current responses produced by 100 μmol.l⁻¹ of NMDA (in the case ofhipocampal neurones), or by 1 mmol.l⁻¹ of glutamate (on recombinant NMDAreceptors) were measured at membrane potential maintained at −60 mV.Similarly as described before, pregnanolone sulfate decreased theamplitude of responses elicited by NMDA. After application of 100_(t)tmol.1⁻ of pregnanolone sulfate the mean inhibition effect reached71.3±5.0% (n=5) on hipocampal neurones, and 67.2±8.2% (n=5) onrecombinant NR1/NR2B receptors (J. Neurosci., 25, 8439-50, 2005). Oursynthetic analogs of pregnanolone sulfate exhibited inhibitory effect(so that the level of inhibition was in the range of 30-70% maximuminhibition). Relative effect of steroid-induced inhibition was used forcalculating IC₅₀. IC₅₀ value was calculated using formulaRI=1−(1/1+([steroid]/IC₅₀)^(h)), where RI denotes relative effect ofsteroid-induced inhibition and h is a parameter of Hill's coefficient(1.2). IC₅₀ values are stated in the following table.

Newly synthesized analogs (8, 18, 19, 21, 22, 34, 35, 40, 49, 50, 51,59, 61, 62, 64, 65, 67, 68, 69, 74, 76, 83, 85, 88, 93, 95, 97, 101,106, 114, 116-124, 126, 127, 128, 130) have the same mechanism of actionat the NMDA receptor as pregnanolone sulfate, but differ in theirrelative affinities for the NMDA receptor (see Table 2).

TABLE 2 Mean % IC₅₀ Concentration Compound change ± SD (μmol) (μmol ·l⁻¹) 3alfa,5beta-Pregnanolone sulfate 67.2 ± 8.2 55 100 ReferenceCompound Pyridinium (2R,4aS,4bS,8aR,10aR)-4a- 78.3 ± 5.5 28.3 100methyltetradecahydrophenanthren-2-yl 2-sulfate (8) Pyridinium(2R,4aS,4bS,7S,8S,8aS,10aR)-7- 60.5 ± 4.6 33 50 (methoxymethyl)-4a,7,8-trimethyltetradecahydrophenanthren-2-yl 2-sulfate (18)4-(((2R,4aS,4bS,7S,8aS,10aR)-7-(Methoxymethyl)-4a,7,8- 47.4 ± 4.3 55 50trimethyltetradecahydrophenanthren-2-yl)oxy)-4-oxobutanoic acid (19)Pyridinium (2R,4aS,7S,8S,10aR)-7-(methoxycarbonyl)- 40.9 ± 6.1 74.6 1004a,7,8-trimethyltetradecahydrophenanthren-2-yl 2-sulfate (22)4-(((2R,4aS,4bS,7R,8aS,10aR)-4a,7- 27.0 ± 10  23.2 10Dimethyltetradecahydrophenanthren-2-yl)oxy)-4- oxobutanoic acid (34)Pyridinium (2R,4aS,4bS,7R,8aS,10aR)-4a,7- 85.0 ± 1.2 12 50dimethyltetradecahydrophenanthren-2-yl 2-sulfate (35) Methyl(2S,4aS,4bS,7R,8aR,10aS)-2,4b-dimethyl-7- 14.4 ± 1.8 224 50(sulfooxy)tetradecahydrophenanthren-2-carboxylate (40) Pyridinium(3R,5R,8S,9S,10S,13S,14S)-10,13- 49.2 ± 6.6 2.1 2dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-3-yl 3-sulfate (49)2-(((3R,5R,8S,9S,10S,13S,14S)-10,13- 64.0 ± 7.0 6.3 10Dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-3-yl)oxy)-2-oxoethanoic acid (50) 2-(((3R,5R,8S,9S,10S,13S,14S)-10,13- 42.0 ± 14.0 15.5 10Dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-3-yl)oxy)-2-oxopropanoic acid (51)2-(((3R,5R,10S,13S,14S)-10,13-Dimethylhexadecahydro-1H- 32.0 ± 5.0 23.210 cyclopenta[a]phenanthren-3-yl)amino)-2-oxoacetic acid (59)((3R,5R,8S,9S,10S,13S,14S)-10,13-Dimethylhexadecahydro- 40.0 ± 5.7 15.410 1H-cyclopenta[a]phenanthren-3-yl)amino)-3-oxopropanoic acid (61)4-(((3R,5R,8S,9S,10S,13S,14S)-10,13- 34.0 ± 5.0 1.7 1Dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-3-yl)oxy)-N,N,N-trimethyl-4-oxobutan-1-amonium chloride (62)4-(((3R,5R,8R,9S,10S,13R,14S)-10,13-Dimethyl-  45.9 ± 11.7 12.9 102,3,4,5,6,7,8,9,10,11,12,13,14,15-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl)oxy)-4-oxobutanoic acid (64)3-(((3R,5R,8R,9S,10S,13R,14S)-10,13-Dimethyl-  44.0 ± 11.0 13.5 102,3,4,5,6,7,8,9,10,11,12,13,14,15-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl)oxy)-3-oxopropanoic acid (65)3-(((3R,5R,8R,10S,13S,14S)-10,13-Dimethyl-17-  53.0 ± 10.2 18.9 20methylenehexadecahydro-1H-cyclopenta[a]phenanthren-3-yl)oxy)-3-oxopropanoic acid (67)4-(((3R,5R,8R,9S,10S,13S,14S)-10,13-Dimethyl-17-  24.2 ± 14.3 18.8 5methylenhexadecahydro-1H-cyclopenta[a]phenanthren-3-yl)oxy)-4-oxobutanoic acid (68)4-(((3R,5R,8R,9S,10S,13S,14S)-10,13-Dimethyl-17- 62.9 ± 5.1 11.6 20methylenhexadecahydro-1H-cyclopenta[a]phenanthren-3-yl)oxy)-4-oxopentanoic acid (69)2-((3R,5R,8R,9S,10S,13S,14S)-10,13-Dimethyl-17- 58.6 ± 9.3 38.7 50oxohexadecahydro-1H-cyclopenta[a]phenanthren-3-yl)acetic acid (74)2-(((3R,5R,8R,9S,10S,13S,14S)-10,13-Dimethyl-17-  50.0 ± 10.0 51.7 50methylenhexadecahydro-1H-cyclopenta[a]phenanthren-3-yl)oxy)-N,N,N-trimethyl-2-oxoethan-1-ammonium chloride (76)3-(((3R,5R,8R,9S,10S,13S,14S,Z)-17-Ethylidene-10,13-  60.5 ± 10.1 20.730 dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-3-yl)oxy)-3-oxopropanoic acid (83)5-(((3R,5R,8R,9S,10S,13S,14S,Z)-17-Ethylidene-10,13- 59.4 ± 7.8 38.4 50dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-3-yl)oxy)-5-oxopentanoic acid (85)3-(((3R,5R,8R,9S,10S,13S,14S,17R)-10,13-Dimethyl-17- 46.0 ± 0.9 11.8 10(prop-1-en-2-yl)hexadecahydro-1H-cyclopenta[a]phenanthren-3-yl)oxy)-3-oxopropanoic acid (88) Pyridinium(3R,5R,8R,9S,10S,13S,14S,17S)-17-iodo-10,13- 90.5 ± 2.2 0.8 5dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-3-yl 3-sulfate (93)Pyridinium (3R,5R,8R,9S,10S,13S,14S)-17,17-difluoro-10,13- 61.2 ± 5.17.0 10 dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-3-yl 3-sulfate(95) Pyridinium (3R,5R,8R,9S,10S,13S,14S,17S)-10,13- 78.0 ± 8.9 45 100dimethylhexadecahydrospiro[cyclopenta-[a]phenanthren- 17,2′-oxiran]-3-yl3-sulfate (97) Pyridinium (2R,4aS,4bS,6aS,10bS,6aS,12aR)-4a,6a- 69.1 ±5.9 2.3 5 dimethyloctadecahydrochrysen-2-yl 2-sulfate (101)(4S)-4-Amino-5-(((2R,4aS,4bS,6aS,10bS,12aR)-4a,6a- 48.7 ± 6.3 10.6 10dimethyloctadecahydrochrysen-2-yl)oxy)-5-oxopentanoic acid (106)Pyridinium (3R,5R,8S,9S,10S,13R,14S)-10,13-dimethyl-16- 59.1 ± 3.8 2.1 3methylenhexadecahydro-1H-cyclopenta[a]phenanthren-3-yl 3-sulfate (114)Pyridinium (3R,5R,8R,9S,10S,13S,14S)-10,13-dimethyl-17- 68.3 ± 4.3 1.4 3methylenehexadecahydro-1H-cyclopenta[a]phenanthren-3-yl 3-sulfate (116)Pyridinium (3R,5R,8S,9S,10S,13R,14S,17S)-10,13,17- 73.1 ± 6.7 1.1 3trimethylhexadecahydro-1H-cyclopenta[a]phenanthren-3-yl 3-sulfate (117)Pyridinium (3R,5R,8S,9S,10S,13S,14R,17R)-10,17- 68.0 ± 7.8 1.5 3dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-3-yl 3-sulfate (118)Pyridinium (3R,5R,8S,9S,10S,13R,14R,17S)-10,17- 81.1 ± 2.1 0.7 3dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-3-yl 3-sulfate (119)Pyridinium (3R,5R,8S,9S,10S,13R,14S,17S)-17-ethyl-10,13-  84.9 ± 3..60.5 3 dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-3-yl 3-sulfate(120) Pyridinium (3R,5R,8R,9S,10S,13S,14S,17R)-10,13-dimethyl- 71.7 ±8.0 0.4 1 17-(prop-1-en-2-yl)hexadecahydro-1H-cyclopenta[a]phenanthren-3-yl 3-sulfate (121) Pyridinium(3R,5R,8R,9S,10S,13R,14S,17R)-17-isopropyl-  59.6 ± 18.8 2.0 310,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren- 3-yl 3-sulfate(122) Pyridinium (3R,5R,8R,9S,10S,13R,14S,17R)-17-((R)-sec- 46.2 ± 2.311.7 10 butyl)-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-3-yl 3-sulfate (123) Pyridinium(3S,3aS,5bR,7aR,9R,11aS,11bS,13aR)-3,11a- 69.1 ± 4.0 51 100dimethylhexadecahydro-1H,3H- naphtho[2′,1′:4,5]indeno[1,7a-c]furan-9-yl9-sulfate (124) Pyridinium (3R,5R,8R,9R,10S,13S,14S)-13- 68.3 ± 7.4 5.410 methylhexadecahydro-1H-cyclopenta[a]phenanthren-3-yl 3- sulfate (126)Pyridinium (3R,5S,8R,9R,10S,13S,14S)-10,13- 34.0 ± 5.0 1.7 3dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-3-yl 3-sulfate (127)Pyridinium (2S,4aR,4bR,8aS,10aS)-4a- 58.0 ± 2.0 36.3 50methyltetradecahydrophenanthren-2-yl 2-sulfate (128)(4S)-4-Amino-5-(((3R,5R,8S,9S,10S,13S,14S)-10,13- 36.7 ± 7.0 1.6 1dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-3-yl)oxy)-5-oxopentanoic acid (130) 1-(3R,5R,8S,9S,10S,13S,14S)-10,13-34.7 ± 7.2 17.7 10Dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-3-yl)-5-oxopyrrolidine-3-carboxylic acid (131) Sodium2-oxo-2-(((3R,5R,8S,9S,10S,13R,14S,17S)- 17.5 ± 1.8 3.7 110,13,17-trimethylhexadecahydro-1H-cyclopenta[a]phenanthren-3-yl)amino)acetate (138)3-Oxo-3-(((3R,5R,8S,9S,10S,13R,14S,17S)-10,13,17- 17.8 ± 6.5 3.9 1trimethylhexadecahydro-1H-cyclopenta[a]phenanthren-3 -yl)amino)propanoic acid (140) Sodium2-(((3R,5R,8R,9S,10S,13R,14S)-17-((R)-sec-butyl)-  7.3 ± 3.0 9.4 110,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-3-yl)amino)-2-oxoacetate (148)3-(((3R,5R,8R,9S,10S,13R,14S)-17-((R)-Sec-butyl)-10,13- 33.6 ± 6.1 18.310 dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-3-yl)amino)-3-oxopropanoic acid (149)2-(((3R,5R,8R,9S,10S,13S,14S,Z)-17-Ethylidene-10,13-  5.7 ± 2.3 11.7 1dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-3-yI)amino)-2-oxoacetic acid (153)3-(((3R,5R,8R,9S,10S,13S,14S,Z)-17-Ethylidene-10,13- 54.4 ± 5.0 8.7 10dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-3-yl)amino)-3-oxopropanoic acid (154)Experiments in vivo

In the Experimental Part 1 were used adults (30-35 g) male laboratorymice, strain CD-1 from Velaz facility, Czech Republic. Mice were housedin plastic boxes with a 12-hour light cycle (lights on at 7:00 pm). Micehad free access to food and water.

In the Experimental Part 2 were used adult (3 months old, 300-400 g)male rat Long-Evans strain.

The animals come from herds Institute of Physiology ASCR. The rats werehoused in clear plastic boxes with the same light cycle as in mice.Animals had free access to food and water. All experiments were carriedout in the light of clay. All experiments were carried out in accordancewith the Law on protection of animals against cruelty.

Used Chemicals

Amphiphilic steroid compounds were dissolved in a solution of 3 g(2-hydroxypropyl)-β-cyclodextrin (CDX, Sigma-Aldrich) and 157 mg ofcitric acid (3-hydroxy-penta-1,3,5-tricarboxylic acid, Sigma-Aldrich) in30 ml of distilled water, the pH was adjusted to 7.4 using sodiumhydroxide (NaOH, Sigma-Aldrich). Thus there were prepared solutions ofthe four steroids for application rates of 0.1, 1, 10 and 100 mg/kg.

The efficacy of the studied compounds was compared to known NMDAantagonist is memantine (Sigma-Aldrich) at a dose of 5 mg/kg in rats,ketamine (Vetoquinol) at 10 mg/kg and dizocilpine (MK-801)(Sigma-Aldrich) at a dose of 0.3 mg/kg in mice. These three compoundswere dissolved in saline (B Braun).

The anesthetized rats were used in operations isoflurane (3.5%, Baxter).To recall excitotoxic lesion of the dorsal hippocampus was applied 0.05mol.l⁻¹ solution of NMDA (Sigma-Aldrich) in 0.4 mol.l⁻¹ phosphate buffersolution prepared by mixing 356 g Na₂HPO₄.12 H₂O (M_(w)358.14) in 4.2 1of distilled water and a solution of 62.4 g NaHPO₄. 2 H₂O (M_(w)156.01)in 0.8 1 of distilled water. The pH of the resulting solution of NMDAwas adjusted to 7.4 with NaOH.

Devices

In operations were used two-arm stereotactic apparatus (KopfInstruments) and microinfusion pump (TSE Systems). The anesthesia wasused vaporizer for isoflurane (AE Services & Supplies) and inhalationmask for rats.

Special apparatus used in behavioral tasks will together with theprocedure in the experiment are described below.

Experimental Part 1

Laboratory mouse was chosen as a model organism for this experiment. Thefollowing experiments were performed as described in Front Behay.Neurosci. 8, 130 (2014).

Primary Behavioral Testing

The purpose of the primary behavioral testing was quickly determined theeffect of compounds on CNS dependent functions and any signs oftoxicity. A simplified modification Irwin test was selected with respectto the behavioral profile of NMDA receptor antagonists. Mice were testedindividually. The study drug was administered intraperitoneally (Table3) at a dose of 1 mg/kg. The control group consisted of individuals thatreceived the CDX solution or saline.

Elevated Plus Maze

The elevated plus maze (EPM) experiment was used to determine the effectof compounds on anxiety of animals at a dose of 1 mg/kg. Two controlgroups of animals were used to which was applied physiological salineand CDX solution, respectively. A noncompetitive NMDA receptorantagonist—ketamine (10 mg/kg) was given to a comparative group. Thecompounds were administered to mice intraperitoneally 30 minutes beforetesting in the EPM.

Open Field

The substances at dose 0.1-100 mg/kg were administered before the testto mice intraperitoneally.

As controls were used intact mice and mice that received CDX solution.Also, MK-801 (0.3 mg/kg) was used for comparison of effects ofnoncompetitive antagonist of NMDA receptors. From the experiment wasevaluated overall track anywhere in the arena as an indicator oflocomotor activity. In addition, it was judged a ten-minute segmentstrack during each experiment. Based on these values, it was possible todetermine the latency time of onset of action and changes in locomotoractivity over time.

In this arrangement, the sedative effect of the studied compounds at thehighest dose was evaluated. Mice were administered the relevantsubstance at dose 100 mg/kg to demonstrate the sedative effect, or viceversa and unexpected toxic effects of high doses of the studiedcompounds. Over time was monitored locomotor activity and possiblechanges characterized by tremor, ataxia, restlessness, or sedation orgeneral anesthesia (absence of response to stimuli, decreased muscletone).

Forced Swimming

This assay was used to monitor the antidepressant effect. Animals float6 minutes in acrylic cylinder in water at 24° C. A period of immobilityis evaluated. The reduction is a manifestation of anti-depressantproperties of drugs.

Passive Avoidance Test

Aversive motivated memory test was evaluated on the basis of latencyinput into the preferred, but unpleasant sensation associated withdelivery device.

Experimental Part 2

In this experiment, the neuroprotective effects were evaluated foramphiphilic steroid compounds. The procedure was performed according toNeuropharmacology 61, 61-68 (2011)

Bilateral Excitotoxic Lession of Dorsal Hippocampus

The rats were randomly divided into eleven groups. The control groupincluded operated animals, which were injected by phosphate buffer pH4.7 into the hippocampus. In the second group, the animals had NMDAlesions of the hippocampus. The animals of the third group were appliedclinically used NMDA antagonist memantine at a dose of 5 mg/kg afterNMDA lesion. The other groups were administered the compounds at a doseof 1 mg/kg .

Alotetické active place avoidance (AAPA)

The test was performed in rotating arena with prohibited sector in theform of a circular sector (60°). If the rat entered the sector, itslimbs received weak electrical impulse. If the animal did not left thesector, the pulse was repeated every 1200 ms.

Test memory and spatial cognition in rats using AAPA wasevaluated overfour sessions, which are in healthy rats sufficient to achieveasymptotic level (Behay. Brain. Res. 189, 139-144 (2008)). To evaluatememory and spatial cognition was used data from the fourth session, andthe number of entries into the forbidden sector and the maximumavoidance sector. These data were analyzed off-line (usingTrackAnalysis, Biosignals Group) and then statistically evaluated. Afterthe experiment, the localization of the lesion was histologicallyverified.

Statistical Evaluation

Data were analyzed using the non parametric test of Maim Whitneycriteria using the GraphPad. The difference was considered significantfor p<0.05, for a non-significant tendency then for 0.05<p<0.075. Thegraph shows averages, error bars represent standard error of the mean(SEM).

Results of In Vivo Experiments Experimental Part 1

The primary behavioral screening did not reveal any abnormal behaviorafter administration of the studied compounds (1 mg/kg), see Table 3.Reflexes of mice and their balance and motor coordination were normal.We did not observed statistically significant differences betweengroups.

The anxiety rate was evaluated based on the number of entries into theopen arms and the total time spent in the open arms of the maze. It hasbeen showed that after administration androstane glutamate at 1 mg/kgsignificantly increased both parameters as compared with both controlgroups (saline and CDX solution, respectively), which demonstrates theanxiolytic effects of androstane glutamate. The highest values of bothmonitored parameters of all the test compounds were achieved afteradministration androstane glutamate at 1 mg/kg.

Application of androstane glutamate at dose 10 mg/kg induced asignificant increase in total time spent in the open arms as compared toanimals of both EPM control groups, number of entries into the open armswas not significantly altered as compared to the control groups (saline,CDX). There was no significant difference between the two control groupsat any of studied parameters.

The antidepressant effect was studied similarly in a forced swimmingtest. The efficacy was evaluated as a decrease flotation in mice afteradministration of the studied compounds, as reference substance was usedketamine, which in accordance with the literature showed antidepressanteffect. Similarly as in the previous screenings was demonstratedsignificant decrease of flotation and longer latency to the firstflotation by androstane glutamate after administration at a dose of 1mg/kg.

Effect of studied compounds on spontaneous locomotor activity of animalswas evaluated by the total path in the open field test over a period of50 minutes. There was not a significant difference among the overallpathway of mice in both control groups (intact animals and CDX).

Administration of dizocilpine (0.3 mg/kg) resulted in significantincreases in overall pathway as compared with the control group that wasadministered with a solution of CDX. There was observed a tendency toincreasement as compared with intact animals (p=0.0653).

Application of substances 67, 68, 69, 81, 84, 106, 124 and 130 at a dose10 and 100 mg/kg resulted in a significant reduction of pathway ascompared with intact mice. At lower doses it was not (unlike groups withdizocilpine) observed hyperlocomotion. These results suggest a low riskof induction of side effects typical for the NMDA antagonist uponadministration of the above mentioned controlled substances. The valuesof pathway in each ten-minute-sections experiment are in directconnection with the changes in locomotor activity over time. Forstatistical comparison of these changes, we used the calculation of areaunder the curve, which is a direct expression of the time course ofchanges in the foregone track. Calculation of the area under the curvewas always done for each observation, and then the resulting data werestatistically compared between the groups.

The results indicate that locomotion of intact animal was graduallyreduced. The trend was similar in the group of mice after theadministration of CDX. There was no significant difference of thelocomotor activity of the intact animals and animals injected with CDXsolution. In the case of dizocilpine (0.3 mg/kg) on the other hand therehas been a gradual increase in locomotor activity. The level oflocomotion (at the highest and relatively stable level) kept between the20th and 50th minutes after administration. The total locomotor activityafter administration of dizocilpine was also significantly higher ascompared with two control groups.

Application of compounds 67, 68, 69, 81, 84, 106, 124 and 130 in thehigher dose of 10 resp. 100 mg/kg induced a significant decrease inlocomotor activity. The slight decrease in locomotion was observedalready after 10 min after administration. Between the 20th and 40thminute, the locomotor activity was minimal, the animals showedsignificant signs of sleepiness and overall sedation, the effect wasmost pronounced for androstan glutamate.

Mild memory impairment in the passive avoidance test was observed onlyfor the substance 84 used in a dose of 1 mg/kg. For other substances, noadverse effects on the formation of memory traces was observed, which isdescribed in the literature for a number of NMDA antagonists.

Experimental Part 2

For reasons of ethical imperative to reduce the number of laboratoryanimals used in the experiment, only the substances with the largestresult were selected to the next phase.

Active Allotetic Place Avoidance:

NMDA lesion of the dorsal hippocampus in rats induced cognitive deficit,manifested as a significant increase in the number of entries into theforbidden areas and a significant reduction of the maximum time avoidingsector in the fourth session of AAPA compared with the control group. Inrats, which were administered after surgery of compound 67 and 130 at adose of 1 mg/kg, there was a significant reduction in the number ofinputs into the forbidden sector during the fourth session of the AAPAgroup compared to NMDA. Both drugs also significantly increased themaximum time avoiding the sector during the fourth session due to NMDA.These findings point to mitigate cognitive deficits and thereforeneuroprotective activity of the compounds 67 and 130. Furthermore, weobserved a tendency to increase the maximum time avoidance and a reducednumber of entries into the forbidden areas in rats with application ofcompounds 68, 81 and 84 and clinically used memantine. Cognitive deficitwas most pronounced in the group of NMDA. Conversely, the best testcognitive results showed controlling animals.

TABLE 3 Summarized Results Sedative Effect NMDA The Rapid Passivelession Onset of Behavioural Avoidance Improvement Forced Sedation TestTest in Cognition Swimming at a Dose No Open Field Test No in AAPA Testof Evidence of No Evidence of Memory after NMDA Reduced Compound 100mg/kg Toxicity Hyperlocomotion Violation Lession Floating 67 + + + ++++68 + + + + +++ 69 + + + −/+ 81 + + + + ++ − 84 + + + −/+ ++ 106 − + + +− − 124 − + + −/+ − − 130 + + + + +++++ + − the desired effect was notobserved, −/+ the desired effect was insufficient, + the desired effectwas observed

INDUSTRIAL APPLICABILITY

The compounds of the present invention are industrially manufacturableand usable for the treatment of many diseases of the central nervoussystem such as: hypoxic and ischemic damage of CNS, stroke and otherpathological changes caused hyperexcitation; neurodegenerative changesand disorders; affective disorders, depression, post-traumatic stressdisorder, and diseases related to stress; schizophrenia and otherpsychotic disorders; pain, hyperalgesia, disturbance in the perceptionof pain; addiction; multiple sclerosis and other autoimmune diseases;epilepsy and other disorders manifesting hyperplazic seizures andchanges in the central nervous system, tumors of the central nervoussystem, including gliomas.

1. An amphiphilic compound with tetradecahydrophenanthrene skeleton ofgeneral formula I,

wherein R¹ is selected from the group comprising (—OSO₃pyH), (—OSO₃Na),(—OSO₃H), NaOOC—R⁶—C(R⁷)—R⁸—, HOOC—R⁶—C(R⁷)—R⁸—, HOOC—Cu (R⁷)—R⁸—, orR⁹—R¹⁰—C(R₁₁)—R¹²—, where R⁶ represents straight or branched C₁ to C₆alkylene or C₂ to C₆ alkenylene chain, unsubstituted or substituted withone or more halogen atoms or amino group or amino group protected byprotecting groups, preferably by tert-butylcarbonyl, or R⁶ meanstrivalent —CH(CH²⁻—)₂ alkylene that forms with the carbon carrying R⁷and with R⁸ being nitrogen a five-membered ring ; R⁷ represents atom ofoxygen, nitrogen or sulphur bound by double bond, or two atoms ofhydrogen, R⁸ represents an at least divalent atom, preferably nitrogen,oxygen or carbon, R⁹ represents a cationic group selected fromguanidinyl derivatives of formula (a),

and quaternary ammonium groups of formula (b)

wherein R¹³ to R²° are selected from hydrogen atoms, linear or branchedC₁ to C₆ alkyl, linear or branched C₂ to C₆ alkenyl, R¹⁰ representsstraight or branched C₁ to C₆ alkylene or C₂ to C₆ alkenylene, whereinthe alkylene and alkenylene are unsubstituted or substituted by 1 to 10halogen atoms or by amino group which is primary or substituted bylinear or branched C₁ to C4 alkyl; R¹¹ represents atom of oxygen,nitrogen or sulphur bound by double bond, or two atoms of hydrogen, andR¹² is selected from the group comprising oxygen, nitrogen and carbonatoms, and when R¹² is carbon or nitrogen, its further valences areoccupied by hydrogen or hydrogens, while any of hydrogen can be replacedby C₁ to C₄ alkyl or C₂ to C₄ alkenyl; R² represents hydrogen atom ormethyl; R³ represents a) hydrogen atom, and then i) R⁴ and R⁵ arehydrogen atoms, or ii) one of R⁴ and R⁵ represents hydrogen atom and theother one represents a straight or branched C₁ to C₆ alkyl or C₂ to C₆alkenyl chain, optionally substituted by 1 to 13 halogen atoms in caseof alkyl and by 1 to 9 halogen atoms in case of alkenyl, or by atom ofoxygen or sulphur bound by a double bond, while one of the methylenegroups in the chain is optionally replaced by oxygen or sulphur atom; orb) straight or branched C₁ to C₆ alkyl or C₂ to C₆ alkenyl, optionallysubstituted by 1 to 13 halogen atoms in case of alkyl or by 1 to 9halogen atoms in case of alkenyl, or by atom of oxygen or sulphur boundby a double bond, while one of the methylene groups in the chain isoptionally replaced by oxygen or sulphur atom, and then R⁴ and R⁵ arehydrogen atoms, or c) C₅ or C₆ alicyclic or aromatic substituent, whilecarbon atoms can be functionalized by 1 to 8 atoms of halogen in case offive-membered alicyclic ring, or 1 to 10 halogen atoms in case ofsix-membered alicyclic ring or by 1 to 4 halogen atoms in case offive-membered aromatic ring or 1 to 5 halogen atoms in case ofsix-membered aromatic ring; and then i) R⁵ is selected from the groupcomprising hydrogen atom, linear or branched C₁ to C6 alkyl, linear orbranched C₂ to C₆ alkenyl, wherein alkyl or alkenyl is optionallysubstituted by 1 to 13 halogen atoms in case of alkyl and by 1 to 9halogen atoms in case of alkenyl, or by double bond bound atom of oxygenor sulphur, while one of the methylene groups in the chain is optionallyreplaced by oxygen or sulphur atom, or ii) R⁴ and R⁵ represent alkyleneor alkenylene substituent —(CH_(m))_(n)—, where n=3-4, m=1-2, formingwith the parent carbon atoms of the skeleton at position 7 and 8saturated or unsaturated 5- or 6-membered cycle, wherein the hydrogenatoms of the alkenylene substituent are optionally substituted at leastby one halogen atom or linear or branched C₁ to C₄ alkyl or C₂ to C₄alkenyl chain, wherein optionally one methylene group of the alkylenesubstituent forming the ring is replaced by carbonyl group and thecarbon atom at the adjoining position is optionally substituted byanother methylene group, or one methylene group of the alkylenesubstituent forming the ring is optionally replaced by oxygen or sulphuratom, while the sulphur atom is optionally functionalized by oxygenatom; or the hydrogens of one methylene group of alkylen substituent areoptionally replaced by —O—CH₂—, thereby forming oxirane ring, d)substituent —CH₂—O—CH(CH₃)—, then together with the first carbon of thealkylene group formed by R⁴ and R5, ^(wherein)R⁴ and R⁵ representalkylene substituent —(CH₂)₃—, forms a saturated and methylatedheretocycle; and enantiomers of compounds of general formula I, with theproviso that compounds wherein R¹ represents HO₂C—R⁶CR⁷—R⁸—, R⁶ is—(CH₂)₂—, R⁷ is oxygen atom bound by double bond and R⁸ is oxygen atom,while R² and R³ are methyl groups, R⁴ and R⁵ together form group—(CH₂)₃— forming with parent carbon atoms of tetradecahydrophenanthreneskeleton at position 7 and 8 a saturated five-membered ring; withabsolute configuration 3R,5S,8S,9S,10S,13S,14S are excluded from formulaI.
 2. The amphiphilic compound of general formula I according to claim1, selected from: pyridinium(2R,4aS,4bS,8aR,10aR)-4a-methyltetradecahydrophenanthren-2-yl 2-sulfate(8), pyridinium (2R,4aS,4bS,7 S,8S,8aS,10aR)-7-(methoxymethyl)-4a,7,8-trimethyltetradecahydrophenanthren-2-yl2-sulfate (18), 4-(((2R,4aS,4bS,7S,8aS,10aR)-7-(methoxymethyl)-4a,7,8-trimethyltetradecahydrophenanthren-2-yl)oxy)-4-oxobutanoicacid (19), pyridinium (2R,4aS,7S, 8S,10aR)-7-(methoxycarbonyl)-4a,7,8-trimethyltetradecahydrophenanthren-2-yl 2-sulfate (22),4-(((2R,4aS,4bS,7R,8aS,10aR)-4a,7-dimethyltetradecahydrophenanthren-2-yl)oxy)-4-oxobutanoicacid (34), pyridinium(2R,4aS,4bS,7R,8aS,10aR)-4a,7-dimethyltetradecahydrophenanthren-2-yl2-sulfate (35), methyl(2S,4aS,4bS,7R,8aR,10aS)-2,4b-dimethyl-7-(sulfooxy)tetradecahydrophenanthren-2-carboxylate(40), pyridinium(3R,5R,8S,9S,10S,13S,14S)-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-3-yl3-sulfate (49),2-(((3R,5R,8S,9S,10S,13S,14S)-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-3-yl)oxy)-2-oxoethanoicacid (50),2-(((3R,5R,8S,9S,10S,13S,14S)-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-3-yl)oxy)-2-oxopropanoicacid (51),2-(((3R,5R,10S,13S,14S)-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-3-yl)amino)-2-oxoaceticacid (59),((3R,5R,8S,9S,10S,13S,14S)-10,13-dimethylhexadecahydro-1H-cyclopenta[a]fenanthren-3-yl)amino)-3-oxopropanoicacid (61),4-(((3R,5R,8S,9S,10S,13S,14S)-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-3-yl)oxy)-N,N,N-trimethyl-4-oxobutan-l-amoniumchloride (62),4-(((3R,5R,8R,9S,10S,13R,14S)-10,13-dimethyl-2,3,4,5,6,7,8,9,10,11,12,13,14,15-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl)oxy)-4-oxobutanoicacid (64),3-(((3R,5R,8R,9S,10S,13R,14S)-10,13-dimethyl-2,3,4,5,6,7,8,9,10,11,12,13,14,15-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl)oxy)-3-oxopropanoicacid (65),3-(((3R,5R,8R,10S,13S,14S)-10,13-dimethyl-17-methylenehexadecahydro-1H-cyclopenta[a]phenanthren-3-yl)oxy)-3-oxopropanoicacid (67),4-(((3R,5R,8R,9S,10S,13S,14S)-10,13-dimethyl-17-methylenhexadecahydro-1H-cyclopenta[a]phenanthren-3-yl)oxy)-4-oxobutanoicacid (68),4-(((3R,5R,8R,9S,10S,13S,14S)-10,13-dimethyl-17-methylenhexadecahydro-1H-cyclopenta[a]phenanthren-3-yl)oxy)-4-oxopentanoicacid (69),2-((3R,5R,8R,9S,10S,13S,14S)-10,13-dimethyl-17-oxohexadecahydro-1H-cyclopentalalphenanthren-3-yl)aceticacid (74),2-(((3R,5R,8R,9S,10S,13S,14S)-10,13-dimethyl-17-methylenehexadecahydro-1H-cyclopenta[a]phenanthren-3-yl)oxy)-N,N,N-trimethyl-2-oxoethan-1-ammoniumchloride (76),3-(((3R,5R,8R,9S,10S,13S,14S,Z)-17-ethylidene-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-3-yl)oxy)-3-oxopropanoicacid (83),5-(((3R,5R,8R,9S,10S,13S,14S,Z)-17-ethylidene-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-3-yl)oxy)-5-oxopentanoicacid (85),3-(((3R,5R,8R,9S,10S,13S,14S,17R)-10,13-dimethyl-17-(prop-1-en-2-yl)hexadecahydro-1H-cyclopenta[a]phenanthren-3-yl)oxy)-3-oxopropanoicacid (88), pyridinium(3R,5R,8R,9S,10S,13S,14S,17S)-17-iodo-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-3-yl3-sulfate (93), pyridinium(3R,5R,8R,9S,10S,13S,14S)-17,17-difluoro-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-3-yl3-sulfate (95), pyridinium (3R,5R,8R,9S,10S,13S,14S,17S)-10,13-dimethylhexadecahydrospirolcyclopenta-[a]phenanthren-17,2′-oxiranel-3-yl3-sulfate (97), pyridinium(2R,4aS,4bS,6aS,10bS,6aS,12aR)-4a,6a-dimethyloctadecahydrochrysen-2-yl2-sulfate (101),(4S)-4-amino-5-(((2R,4aS,4bS,6aS,10bS,12aR)-4a,6a-dimethyloctadecahydrochrysen-2-yl)oxy)-5-oxopentanoicacid (106), pyridinium(3R,5R,8S,9S,10S,13R,14S)-10,13-dimethyl-16-methylenehexadecahydro-1H-cyclopenta[a]phenanthren-3-yl3-sulfate (114), pyridinium(3R,5R,8R,9S,10S,13S,14S)-10,13-dimethyl-17-methylenehexadecahydro-1H-cyclopenta[a]phenanthren-3-yl3-sulfate (116), pyridinium(3R,5R,8S,9S,10S,13R,14S,17S)-10,13,17-trimethylhexadecahydro-1H-cyclopenta[a]phenanthren-3-yl3-sulfate (117), pyridinium(3R,5R,8S,9S,10S,13S,14R,17R)-10,17-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-3-yl3-sulfate (118), pyridinium(3R,5R,8S,9S,10S,13R,14R,17S)-10,17-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-3-yl3-sulfate (119), pyridinium(3R,5R,8S,9S,10S,13R,14S,17S)-17-ethyl-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-3-yl3-sulfate (120), pyridinium(3R,5R,8R,9S,10S,13S,14S,17R)-10,13-dimethyl-17-(prop-1-en-2-yl)hexadecahydro-1H-cyclopenta[a]phenanthren-3-yl3-sulfate (121), pyridinium(3R,5R,8R,9S,10S,13R,14S,17R)-17-isopropyl-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-3-yl3-sulfate (122), pyridinium(3R,5R,8R,9S,10S,13R,14S,17R)-174(R)-sec-butyl)-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-3-yl3-sulfate (123), pyridinium(3S,3aS,5bR,7aR,9R,11aS,11bS,13aR)-3,11a-dimethylhexadecahydro-1H,3H-naphthol[2′,1′:4,5]indeno[1,7a-c]furan-9-yl9-sulfate (124), pyridinium(3R,5R,8R,9R,10S,13S,14S)-13-methylhexadecahydro-1H-cyclopenta[a]phenanthren-3-yl3-sulfate (126), pyridinium(3R,5S,8R,9R,10S,13S,14S)-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-3-yl 3-sulfate (127), pyridinium(2S,4aR,4bR,8aS,10aS)-4a-methyltetradecahydrophenanthren-2-yl 2-sulfate(128),(4S)-4-amino-5-(((3R,5R,8S,9S,10S,13S,14S)-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-3-yl)oxy)-5-oxopentanoicacid (130),1-((3R,5R,8S,9S,10S,13S,14S)-10,13-Dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-3-yl)-5-oxopyrrolidine-3-carboxylicacid (131), mixture of isomers sodium2-oxo-2-(((3R,5R,8S,9S,10S,13R,14S,17S)-10,13,17-trimethylhexadecahydro-1H-cyclopenta[a]phenanthren-3-yl)amino)acetate(138),3-oxo-3-(((3R,5R,8S,9S,10S,13R,14S,17S)-10,13,17-trimethylhexadecahydro-1H-cyclopenta[a]phenanthren-3-yl)amino)propanoicacid (140), sodium2-(((3R,5R,8R,9S,10S,13R,14S)-174(R)-sec-butyl)-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-3-yl)amino)-2-oxoacetate(148),3-(((3R,5R,8R,9S,10S,13R,14S)-17-((R)-sec-butyl)-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-3-yl)amino)-3-oxopropanoicacid (149),2-(((3R,5R,8R,9S,10S,13S,14S,Z)-17-ethylidene-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-3-yl)amino)-2-oxoaceticacid (154),3-(((3R,5R,8R,9S,10S,13S,14S,Z)-17-ethylidene-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-3-yl)amino)-3-oxopropanoicacid (155).
 3. The amphiphilic compound with tetradecahydrophenanthreneskeleton of general formula I according to claim 1 for use as amedicament.
 4. The amphiphilic compound with tetradecahydrophenanthreneskeleton of general formula I according to claim 1 for use in a methodof treatment of neuropsychiatric disorders associated with imbalance inglutamatergic neurotransmitter system, such as ischemic damage of CNS,neurodegenerative changes and disorders of CNS, affective disorders,depression, post-traumatic stress disorder, and diseases related tostress, anxiety, schizophrenia and psychotic disorders, pain, addiction,multiple sclerosis, epilepsy, glioma.
 5. A pharmaceutical compositionfor human or veterinary use, characterized in that it comprises asactive ingredient at least one amphiphilic compound withtetradecahydrophenanthrene skeleton of general formula I according toclaim
 1. 6. The pharmaceutical composition according to claim 5, for usein a method of treatment of neuropsychiatric disorders associated withan imbalance in glutamatergic neurotransmitter system, such as ischemicdamage of CNS, neurodegenerative changes and disorders of CNS, affectivedisorders, depression, post-traumatic stress disorder, and diseasesrelated to stress, anxiety, schizophrenia and psychotic disorders, pain,addiction, multiple sclerosis, epilepsy, glioma.
 7. Use of theamphiphilic compound with tetradecahydrophenanthrene skeleton of generalformula I according to claim 1, for the manufacture of veterinary andhuman pharmaceutical composition for treating neuropsychiatric disordersassociated with imbalance of glutamatergic neurotransmitter system, suchas ischemic damage to the central nervous system, neurodegenerativechanges and disorders of the central nervous system, affectivedisorders, depression, post-traumatic stress disorder, and diseasesrelated to stress, anxiety, schizophrenia and psychotic disorders, pain,addiction, multiple sclerosis, epilepsy, glioma.
 8. Use of anamphiphilic compound with tetradecahydrophenanthrene skeleton of generalformula I according to claim 1, for manufacturing standards ofneuroprotective agents, antidepressants, antianxiety, mood stabilizers,hypnotives, sedatives, analgesics, anesthetics, antipsychotics,neuroleptics and procognitives or analytical standards used inexperimental research and analytical chemistry.
 9. Use of an amphiphiliccompound with tetradecahydrophenanthrene skeleton of general formula Iaccording to claim 1, as food additives or active ingredients ofcosmetic preparations intended for improving the response of theindividual parts of the organism to increased stress, in particular toincreased oxidative stress, nutritional stress and stress caused by freeradicals, or to aging.